METHODS FOR QUANTITATING SMALL RNA MOLECULES

- MERCK SHARP & DOHME CORP.

In one aspect, the present invention provides methods for amplifying a microRNA molecule to produce DNA molecules. The methods each include the steps of: (a) using primer extension to make a DNA molecule that is complementary to a target microRNA molecule; and (b) using a universal forward primer and a reverse primer to amplify the DNA molecule to produce amplified DNA molecules. In some embodiments of the method, at least one of the forward primer and the reverse primer comprise at least one locked nucleic acid molecule.

Skip to: Description  ·  Claims  · Patent History  ·  Patent History
Description
CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 11/779,759, filed Jul. 18, 2007, which is a continuation-in-part of application Ser. No. 10/579,029, filed Nov. 19, 2008, which is the National Stage of International Application No. PCT/US2006/002591, filed Jan. 25, 2006, which claims the benefit of Provisional Application No. 60/647,178, filed Jan. 25, 2005, all of which are incorporated herein by reference in their entirety.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is 37790_Sequence_Final.txt. The text file is 250 KB; was created on Sep. 20, 2011; and is being submitted via EFS-Web with the filing of the specification.

FIELD OF THE INVENTION

The present invention relates to methods of amplifying and quantitating small RNA molecules.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is an evolutionarily conserved process that functions to inhibit gene expression (Bernstein et al. (2001), Nature 409:363-6; Dykxhoorn et al. (2003) Nat. Rev. Mol. Cell. Biol. 4:457-67). The phenomenon of RNAi was first described in Caenorhabditis elegans, where injection of double-stranded RNA (dsRNA) led to efficient sequence-specific gene silencing of the mRNA that was complementary to the dsRNA (Fire et al. (1998) Nature 391:806-11). RNAi has also been described in plants as a phenomenon called post-transcriptional gene silencing (PTGS), which is likely used as a viral defense mechanism (Jorgensen (1990) Trends Biotechnol. 8:340-4; Brigneti et al. (1998) EMBO J. 17:6739-46; Hamilton & Baulcombe (1999) Science 286:950-2).

An early indication that the molecules that regulate PTGS were short RNAs processed from longer dsRNA was the identification of short 21 to 22 nucleotide dsRNA derived from the longer dsRNA in plants (Hamilton & Baulcombe (1999) Science 286:950-2). This observation was repeated in Drosophila embryo extracts where long dsRNA was found processed into 21-25 nucleotide short RNA by the RNase III type enzyme, Dicer (Elbashir et al. (2001) Nature 411:494-8; Elbashir et al. (2001) EMBO J. 20:6877-88; Elbashir et al. (2001) Genes Dev. 15:188-200). These observations led Elbashir et al. to test if synthetic 21-25 nucleotide synthetic dsRNAs function to specifically inhibit gene expression in Drosophila embryo lysates and mammalian cell culture (Elbashir et al. (2001) Nature 411:494-8; Elbashir et al. (2001) EMBO J. 20:6877-88; Elbashir et al. (2001) Genes Dev. 15:188-200). They demonstrated that small interfering RNAs (siRNAs) had the ability to specifically inhibit gene expression in mammalian cell culture without induction of the interferon response.

These observations led to the development of techniques for the reduction, or elimination, of expression of specific genes in mammalian cell culture, such as plasmid-based systems that generate hairpin siRNAs (Brummelkamp et al. (2002) Science 296:550-3; Paddison et al. (2002) Genes Dev. 16:948-58; Paddison et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:1443-8; Paul et al. 2002) Nat. Biotechnol. 20:404-8). siRNA molecules can also be introduced into cells, in vivo, to inhibit the expression of specific proteins (see, e.g., Soutschek, J., et al., Nature 432 (7014):173-178 (2004)).

siRNA molecules have promise both as therapeutic agents for inhibiting the expression of specific proteins, and as targets for drugs that affect the activity of siRNA molecules that function to regulate the expression of proteins involved in a disease state. A first step in developing such therapeutic agents is to measure the amounts of specific siRNA molecules in different cell types within an organism, and thereby construct an “atlas” of siRNA expression within the body. Additionally, it will be useful to measure changes in the amount of specific siRNA molecules in specific cell types in response to a defined stimulus, or in a disease state.

Short RNA molecules are difficult to quantitate. For example, with respect to the use of PCR to amplify and measure the small RNA molecules, most PCR primers are longer than the small RNA molecules, and so it is difficult to design a primer that has significant overlap with a small RNA molecule, and that selectively hybridizes to the small RNA molecule at the temperatures used for primer extension and PCR amplification reactions.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for amplifying a microRNA molecule to produce cDNA molecules. The methods include the steps of: (a) producing a first DNA molecule that is complementary to a target microRNA molecule using primer extension; and (b) amplifying the first DNA molecule to produce amplified DNA molecules using a universal forward primer and a reverse primer. In some embodiments of the method, at least one of the forward primer and the reverse primer comprise at least one locked nucleic acid molecule. It will be understood that, in the practice of the present invention, typically numerous (e.g., millions) of individual microRNA molecules are amplified in a sample (e.g., a solution of RNA molecules isolated from living cells).

In another aspect, the present invention provides methods for measuring the amount of a target microRNA in a sample from a living organism. The methods of this aspect of the invention include the step of measuring the amount of a target microRNA molecule in a multiplicity of different cell types within a living organism, wherein the amount of the target microRNA molecule is measured by a method including the steps of: (1) producing a first DNA molecule complementary to the target microRNA molecule in the sample using primer extension; (2) amplifying the first DNA molecule to produce amplified DNA molecules using a universal forward primer and a reverse primer; and (3) measuring the amount of the amplified DNA molecules. In some embodiments of the method, at least one of the forward primer and the reverse primer comprise at least one locked nucleic acid molecule.

In another aspect, the invention provides nucleic acid primer molecules consisting of sequence SEQ ID NO:1 to SEQ ID NO: 499, as shown in TABLE 1, TABLE 2, TABLE 6, and TABLE 7. The primer molecules of the invention can be used as primers for detecting mammalian microRNA target molecules, using the methods of the invention described herein.

In another aspect, the present invention provides kits for detecting at least one mammalian target microRNA, the kits comprising one or more primer sets specific for the detection of a target microRNA, each primer set comprising (1) an extension primer for producing a cDNA molecule complementary to a target microRNA, (2) a universal forward PCR primer for amplifying the cDNA molecule and (3) a reverse PCR primer for amplifying the cDNA molecule. The extension primer comprises a first portion that hybridizes to the target microRNA molecule and a second portion that includes a hybridization sequence for a universal forward PCR primer. The reverse PCR primer comprises a sequence selected to hybridize to a portion of the cDNA molecule. In some embodiments of the kit, at least one of the universal forward and reverse primers include at least one locked nucleic acid molecule. The kits of the invention may be used to practice various embodiments of the methods of the invention.

The present invention is useful, for example, for quantitating specific microRNA molecules within different types of cells in a living organism, or, for example, for measuring changes in the amount of specific microRNAs in living cells in response to a stimulus (e.g., in response to administration of a drug).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIG. 1 shows a flow chart of a representative method of the present invention;

FIG. 2 graphically illustrates the standard curves for assays specific for the detection of microRNA targets miR-95 and miR-424 as described in EXAMPLE 3;

FIG. 3A is a histogram plot showing the expression profile of miR-1 across a panel of total RNA isolated from twelve tissues as described in EXAMPLE 5;

FIG. 3B is a histogram plot showing the expression profile of miR-124 across a panel of total RNA isolated from twelve tissues as described in EXAMPLE 5; and

FIG. 3C is a histogram plot showing the expression profile of miR-150 across a panel of total RNA isolated from twelve tissues as described in EXAMPLE 5.

 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with the foregoing, in one aspect, the present invention provides methods for amplifying a microRNA molecule to produce cDNA molecules. The methods include the steps of: (a) using primer extension to make a DNA molecule that is complementary to a target microRNA molecule; and (b) using a universal forward primer and a reverse primer to amplify the DNA molecule to produce amplified DNA molecules. In some embodiments of the method, at least one of the universal forward primer and the reverse primer comprises at least one locked nucleic acid molecule.

As used herein, the term “locked nucleic acid molecule” (abbreviated as LNA molecule) refers to a nucleic acid molecule that includes a 2′-0,4′-C-methylene-β-D-ribofuranosyl moiety. Exemplary 2′-0,4′-C-methylene-β-D-ribofuranosyl moieties, and exemplary LNAs including such moieties, are described, for example, in Petersen, M., and Wengel, J., Trends in Biotechnology 21(2):74-81 (2003) which publication is incorporated herein by reference in its entirety.

As used herein, the term “microRNA” refers to an RNA molecule that has a length in the range of from 21 nucleotides to 25 nucleotides. Some microRNA molecules (e.g., siRNA molecules) function in living cells to regulate gene expression.

Representative Method of the Invention. FIG. 1 shows a flowchart of a representative method of the present invention. In the method represented in FIG. 1, a microRNA is the template for synthesis of a complementary first DNA molecule. The synthesis of the first DNA molecule is primed by an extension primer, and so the first DNA molecule includes the extension primer and newly synthesized DNA (represented by a dotted line in FIG. 1). The synthesis of DNA is catalyzed by reverse transcriptase.

The extension primer includes a first portion (abbreviated as FP in FIG. 1) and a second portion (abbreviated as SP in FIG. 1). The first portion hybridizes to the microRNA target template, and the second portion includes a nucleic acid sequence that hybridizes with a universal forward primer, as described infra.

A quantitative polymerase chain reaction is used to make a second DNA molecule that is complementary to the first DNA molecule. The synthesis of the second DNA molecule is primed by the reverse primer that has a sequence that is selected to specifically hybridize to a portion of the target first DNA molecule. Thus, the reverse primer does not hybridize to nucleic acid molecules other than the first DNA molecule. The reverse primer may optionally include at least one LNA molecule located within the portion of the reverse primer that does not overlap with the extension primer. In FIG. 1, the LNA molecules are represented by shaded ovals.

A universal forward primer hybridizes to the 3′ end of the second DNA molecule and primes synthesis of a third DNA molecule. It will be understood that, although a single microRNA molecule, single first DNA molecule, single second DNA molecule, single third DNA molecule and single extension, forward and reverse primers are shown in FIG. 1, typically the practice of the present invention uses reaction mixtures that include numerous copies (e.g., millions of copies) of each of the foregoing nucleic acid molecules.

The steps of the methods of the present invention are now considered in more detail.

Preparation of microRNA Molecules Useful as Templates. microRNA molecules useful as templates in the methods of the invention can be isolated from any organism (e.g., eukaryote, such as a mammal) or part thereof, including organs, tissues, and/or individual cells (including cultured cells). Any suitable RNA preparation that includes microRNAs can be used, such as total cellular RNA.

RNA may be isolated from cells by procedures that involve lysis of the cells and denaturation of the proteins contained therein. Cells of interest include wild-type cells, drug-exposed wild-type cells, modified cells, and drug-exposed modified cells.

Additional steps may be employed to remove some or all of the DNA. Cell lysis may be accomplished with a nonionic detergent, followed by microcentrifugation to remove the nuclei and hence the bulk of the cellular DNA. In one embodiment, RNA is extracted from cells of the various types of interest using guanidinium thiocyanate lysis followed by CsCl centrifugation to separate the RNA from DNA (see, Chirgwin et al., 1979, Biochemistry 18:5294-5299). Separation of RNA from DNA can also be accomplished by organic extraction, for example, with hot phenol or phenol/chloroform/isoamyl alcohol.

If desired, RNase inhibitors may be added to the lysis buffer. Likewise, for certain cell types, it may be desirable to add a protein denaturation/digestion step to the protocol.

The sample of RNA can comprise a multiplicity of different microRNA molecules, each different microRNA molecule having a different nucleotide sequence. In a specific embodiment, the microRNA molecules in the RNA sample comprise at least 100 different nucleotide sequences. In other embodiments, the microRNA molecules of the RNA sample comprise at least 500, 1,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000 90,000, or 100,000 different nucleotide sequences.

The methods of the invention may be used to detect the presence of any microRNA. For example, the methods of the invention can be used to detect one or more of the microRNA targets described in a database such as “the miRBase sequence database” as described in Griffith-Jones et al. (2004), Nucleic Acids Research 32:D109-D111, and Griffith-Jones et al. (2006), Nucleic Acids Research 34:D140-D144, which is publicly accessible on the World Wide Web at the Wellcome Trust Sanger Institute website at http://microrna.sanger.ac.uk/sequences/. A list of exemplary microRNA targets is also described in the following references: Lagos-Quintana et al., Curr. Biol. 12(9):735-9 (2002).

Synthesis of DNA Molecules Using microRNA Molecules As Templates. In the practice of the methods of the invention, first DNA molecules are synthesized that are complementary to the microRNA target molecules, and that are composed of an extension primer and newly synthesized DNA (wherein the extension primer primes the synthesis of the newly synthesized DNA). Individual first DNA molecules can be complementary to a whole microRNA target molecule, or to a portion thereof; although typically an individual first DNA molecule is complementary to a whole microRNA target molecule. Thus, in the practice of the methods of the invention, a population of first DNA molecules is synthesized that includes individual DNA molecules that are each complementary to all, or to a portion, of a target microRNA molecule.

The synthesis of the first DNA molecules is catalyzed by reverse transcriptase. Any reverse transcriptase molecule can be used to synthesize the first DNA molecules, such as those derived from Moloney murine leukemia virus (MMLV-RT), avian myeloblastosis virus (AMV-RT), bovine leukemia virus (BLV-RT), Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV-RT). A reverse transcriptase lacking RNaseH activity (e.g., SUPERSCRIPT III™ sold by Invitrogen, 1600 Faraday Avenue, P.O. Box 6482, Carlsbad, Calif. 92008) is preferred in order to minimize the amount of double-stranded cDNA synthesized at this stage. The reverse transcriptase molecule should also preferably be thermostable so that the DNA synthesis reaction can be conducted at as high a temperature as possible, while still permitting hybridization of primer to the microRNA target molecules.

Priming the Synthesis of the First DNA Molecules. The synthesis of the first DNA molecules is primed using an extension primer. Typically, the length of the extension primer is in the range of from 10 nucleotides to 100 nucleotides, such as 20 to 35 nucleotides. The nucleic acid sequence of the extension primer is incorporated into the sequence of each, synthesized, DNA molecule. The extension primer includes a first portion that hybridizes to a portion of the microRNA molecule. Typically the first portion of the extension primer includes the 3′-end of the extension primer. The first portion of the extension primer typically has a length in the range of from 6 nucleotides to 20 nucleotides, such as from 10 nucleotides to 12 nucleotides. In some embodiments, the first portion of the extension primer has a length in the range of from 3 nucleotides to 25 nucleotides.

The extension primer also includes a second portion that typically has a length of from 18 to 25 nucleotides. For example, the second portion of the extension primer can be 20 nucleotides long. The second portion of the extension primer is located 5′ to the first portion of the extension primer. The second portion of the extension primer includes at least a portion of the hybridization site for the universal forward primer. For example, the second portion of the extension primer can include all of the hybridization site for the universal forward primer, or, for example, can include as little as a single nucleotide of the hybridization site for the universal forward primer (the remaining portion of the hybridization site for the forward primer can, for example, be located in the first portion of the extension primer). An exemplary nucleic acid sequence of a second portion of an extension primer is 5′ CATGATCAGCTGGGCCAAGA 3′ (SEQ ID NO:1).

Amplification of the DNA Molecules. In the practice of the methods of the invention, the first DNA molecules are enzymatically amplified using the polymerase chain reaction. A universal forward primer and a reverse primer are used to prime the polymerase chain reaction. The reverse primer includes a nucleic acid sequence that is selected to specifically hybridize to a portion of a first DNA molecule.

The reverse primer typically has a length in the range of from 10 nucleotides to 100 nucleotides. In some embodiments, the reverse primer has a length in the range of from 12 nucleotides to 20 nucleotides. The nucleotide sequence of the reverse primer is selected to hybridize to a specific target nucleotide sequence under defined hybridization conditions. The reverse primer and extension primer are both present in the PCR reaction mixture, and so the reverse primer should be sufficiently long so that the melting temperature (Tm) is at least 50° C., but should not be so long that there is extensive overlap with the extension primer which may cause the formation of “primer dimers.” “Primer dimers” are formed when the reverse primer hybridizes to the extension primer, and uses the extension primer as a substrate for DNA synthesis, and the extension primer hybridizes to the reverse primer, and uses the reverse primer as a substrate for DNA synthesis. To avoid the formation of “primer dimers,” typically the reverse primer and the extension primer are designed so that they do not overlap with each other by more than 6 nucleotides. If it is not possible to make a reverse primer having a Tm of at least 50° C., and wherein the reverse primer and the extension primer do not overlap by more than 6 nucleotides, then it is preferable to lengthen the reverse primer (since Tm usually increases with increasing oligonucleotide length) and decrease the length of the extension primer.

The reverse primer primes the synthesis of a second DNA molecule that is complementary to the first DNA molecule. The universal forward primer hybridizes to the portion of the second DNA molecule that is complementary to the second portion of the extension primer which is incorporated into all of the first DNA molecules. The universal forward primer primes the synthesis of third DNA molecules. The universal forward primer typically has a length in the range of from 16 nucleotides to 100 nucleotides. In some embodiments, the universal forward primer has a length in the range of from 16 nucleotides to 30 nucleotides. The universal forward primer may include at least one locked nucleic acid molecule. In some embodiments, the universal forward primer includes from 1 to 25 locked nucleic acid molecules. The nucleic acid sequence of an exemplary universal forward primer is set forth in SEQ ID NO:13.

In general, the greater the number of amplification cycles during the polymerase chain reaction, the greater the amount of amplified DNA that is obtained. On the other hand, too many amplification cycles (e.g., more than 35 amplification cycles) may result in spurious and unintended amplification of non-target double-stranded DNA. Thus, in some embodiments, a desirable number of amplification cycles is between one and 45 amplification cycles, such as from one to 25 amplification cycles, or such as from five to 15 amplification cycles, or such as ten amplification cycles.

Use of LNA Molecules and Selection of Primer Hybridization Conditions. Hybridization conditions are selected that promote the specific hybridization of a primer molecule to the complementary sequence on a substrate molecule. With respect to the hybridization of a 12 nucleotide first portion of an extension primer to a microRNA, it has been found that specific hybridization occurs at a temperature of 50° C. Similarly, it has been found that hybridization of a 20 nucleotide universal forward primer to a complementary DNA molecule, and hybridization of a reverse primer (having a length in the range of from 12-20 nucleotides, such as from 14-16 nucleotides) to a complementary DNA molecule occurs at a temperature of 50° C. By way of example, it is often desirable to design extension, reverse and universal forward primers that each have a hybridization temperature in the range of from 50° C. to 60° C.

In some embodiments, LNA molecules can be incorporated into at least one of the extension primer, reverse primer, and universal forward primer to raise the Tm of one, or more, of the foregoing primers to at least 50° C. Incorporation of an LNA molecule into the portion of the reverse primer that hybridizes to the target first DNA molecule, but not to the extension primer, may be useful because this portion of the reverse primer is typically no more than 10 nucleotides in length. For example, the portion of the reverse primer that hybridizes to the target first DNA molecule, but not to the extension primer, may include at least one locked nucleic acid molecule (e.g., from 1 to 25 locked nucleic acid molecules). In some embodiments, two or three locked nucleic acid molecules are included within the first 8 nucleotides from the 5′ end of the reverse primer.

The number of LNA residues that must be incorporated into a specific primer to raise the Tm to a desired temperature mainly depends on the length of the primer and the nucleotide composition of the primer. A tool for determining the effect on Tm of one or more LNAs in a primer is available on the Internet Web site of Exiqon, Bygstubben 9, DK-2950 Vedbaek, Denmark.

Although one or more LNAs can be included in any of the primers used in the practice of the present invention, it has been found that the efficiency of synthesis of cDNA is low if an LNA is incorporated into the extension primer. While not wishing to be bound by theory, LNAs may inhibit the activity of reverse transcriptase.

Detecting and Measuring the Amount of the Amplified DNA Molecules. The amplified DNA molecules can be detected and quantitated by the presence of detectable marker molecules, such as fluorescent molecules. For example, the amplified DNA molecules can be detected and quantitated by the presence of a dye (e.g., SYBR green) that preferentially or exclusively binds to double stranded DNA during the PCR amplification step of the methods of the present invention. For example, Molecular Probes, Inc. (29851 Willow Creek Road, Eugene, Oreg. 97402) sells quantitative PCR reaction mixtures that include SYBR green dye. By way of further example, another dye (referred to as “BEBO”) that can be used to label double stranded DNA produced during real-time PCR is described by Bengtsson, M., et al., Nucleic Acids Research 3/(8):e45 (Apr. 15, 2003), which publication is incorporated herein by reference. Again by way of example, a forward and/or reverse primer that includes a fluorophore and quencher can be used to prime the PCR amplification step of the methods of the present invention. The physical separation of the fluorophore and quencher that occurs after extension of the labeled primer during PCR permits the fluorophore to fluoresce, and the fluorescence can be used to measure the amount of the PCR amplification products. Examples of commercially available primers that include a fluorophore and quencher include Scorpion primers and Uniprimers, which are both sold by Molecular Probes, Inc.

Representative Uses of the Present Invention. The present invention is useful for producing cDNA molecules from microRNA target molecules. The amount of the DNA molecules can be measured which provides a measurement of the amount of target microRNA molecules in the starting material. For example, the methods of the present invention can be used to measure the amount of specific microRNA molecules (e.g., specific siRNA molecules) in living cells. Again by way of example, the present invention can be used to measure the amount of specific microRNA molecules (e.g., specific siRNA molecules) in different cell types in a living body, thereby producing an “atlas” of the distribution of specific microRNA molecules within the body. Again by way of example, the present invention can be used to measure changes in the amount of specific microRNA molecules (e.g., specific siRNA molecules) in response to a stimulus, such as in response to treatment of a population of living cells with a drug.

Thus, in another aspect, the present invention provides methods for measuring the amount of a target microRNA in a multiplicity of different cell types within a living organism (e.g., to make a microRNA “atlas” of the organism). The methods of this aspect of the invention each include the step of measuring the amount of a target microRNA molecule in a multiplicity of different cell types within a living organism, wherein the amount of the target microRNA molecule is measured by a method comprising the steps of: (1) using primer extension to make a DNA molecule complementary to the target microRNA molecule isolated from a cell type of a living organism; (2) using a universal forward primer and a reverse primer to amplify the DNA molecule to produce amplified DNA molecules, and (3) measuring the amount of the amplified DNA molecules. In some embodiments of the methods, at least one of the forward primer and the reverse primer comprises at least one locked nucleic acid molecule. The measured amounts of amplified DNA molecules can, for example, be stored in an interrogatable database in electronic form, such as on a computer-readable medium (e.g., a floppy disc).

In some embodiments, the methods may be used to discriminate between two or more mammalian target microRNA that have a similar sequence in a sample from a living organism, the method comprising the steps of: (a) producing a first DNA molecule that is complementary to the first microRNA molecule using a first extension primer specific to the first microRNA molecule; (b) amplifying the first DNA molecule to produce a first population of amplified DNA molecules using a universal forward primer and a first reverse primer; (c) producing a second DNA molecule that is complementary to the second microRNA molecule using a second extension primer specific to the second microRNA molecule; (d) amplifying the second DNA molecule to produce a second population of amplified DNA molecules using a universal forward primer and a second reverse primer; (e) measuring the amount of the first and second population of amplified DNA molecules, wherein the first and second extension primers or the first and second reverse primers differ by one or more nucleotides in the portion that is complementary to the target microRNA. This method may be used to discriminate between microRNA targets that differ by one, two, three or more nucleotides, by designing the gene-specific region of the first and second extension primers to hybridize to the region of the microRNA targets that are not identical.

In another aspect, the invention provides nucleic acid primer molecules consisting of sequence SEQ ID NO:1 to SEQ ID NO: 499, as shown in TABLE 1, TABLE 2, TABLE 6, and TABLE 7. The primer molecules of the invention can be used as primers for detecting mammalian microRNA target molecules, using the methods of the invention described herein.

In another aspect, the invention provides sets of nucleic acid primers consisting of SEQ ID NO:500 to SEQ ID NO: 965, as shown in TABLE 8. The sets of primer molecules of the invention can be used for the detection of microRNA target molecules from human, mouse, and rat, using the methods of the invention described herein.

In another aspect, the present invention provides kits for detecting at least one mammalian target microRNA, the kits comprising one or more primer sets specific for the detection of a target microRNA, each primer set comprising (1) an extension primer for producing a cDNA molecule complementary to a target microRNA, (2) a universal forward PCR primer, and (3) a reverse PCR primer for amplifying the cDNA molecule. The extension primer comprises a first portion that hybridizes to the target microRNA molecule and a second portion that includes a hybridization sequence for a universal forward PCR primer. The reverse PCR primer comprises a sequence selected to hybridize to a portion of the cDNA molecule. In some embodiments of the kits, at least one of the universal forward and reverse primers includes at least one locked nucleic acid molecule.

The extension primer, universal forward and reverse primers for inclusion in the kit may be designed to detect any mammalian target microRNA in accordance with the methods described herein. Nonlimiting examples of human target microRNA target molecules and exemplary target-specific extension primers and reverse primers are listed below in TABLE 1, TABLE 2, and TABLE 6. Nonlimiting examples of murine target microRNA target molecules and exemplary target-specific extension primers and reverse primers are listed below in TABLE 7. A nonlimiting example of a universal forward primer is set forth as SEQ ID NO: 13.

In certain embodiments, the kit includes a set of primers comprising an extension primer, reverse and universal forward primers for a selected target microRNA molecule that each have a hybridization temperature in the range of from 50° C. to 60° C.

In certain embodiments, the kit includes a plurality of primer sets that may be used to detect a plurality of mammalian microRNA targets, such as two microRNA targets up to several hundred microRNA targets.

In certain embodiments, the kit comprises one or more primer sets capable of detecting at least one or more of the following human microRNA target templates: of miR-1, miR-7, miR-9*, miR-10a, miR-10b, miR-15a, miR-15b, miR-16, miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, miR-21, miR-22, miR-23a, miR-23b, miR-24, miR-25, miR-26a, miR-26b, miR-27a, miR-28, miR-29a, miR-29b, miR-29c, miR-30a-5p, miR-30b, miR-30c, miR-30d, miR-30e-5p, miR-30e-3p, miR-31, miR-32, miR-33, miR-34a, miR-34b, miR-34c, miR-92, miR-93, miR-95, miR-96, miR-98, miR-99a, miR-99b, miR-100, miR-101, miR-103, miR-105, miR-106a, miR-107, miR-122, miR-122a, miR-124, miR-124, miR-124a, miR-125 a, miR-125b, miR-126, miR-126*, miR-127, miR-128a, miR-128b, miR-129, miR-130a, miR-130b, miR-132, miR-133a, miR-133b, miR-134, miR-135a, miR-135b, miR-136, miR-137, miR-138, miR-139, miR-140, miR-141, miR-142-3p, miR-143, miR-144, miR-145, miR-146, miR-147, miR-148a, miR-148b, miR-149, miR-150, miR-151, miR-152, miR-153, miR-154*, miR-154, miR-155, miR-181a, miR-181b, miR-181c, miR-182*, miR-182, miR-183, miR-184, miR-185, miR-186, miR-187, miR-188, miR-189, miR-190, miR-191, miR-192, miR-193, miR-194, miR-195, miR-196a, miR-196b, miR-197, miR-198, miR-199a*, miR-199a, miR-199b, miR-200a, miR-200b, miR-200c, miR-202, miR-203, miR-204, miR-205, miR-206, miR-208, miR-210, miR-211, miR-212, miR-213, miR-213, miR-214, miR-215, miR-216, miR-217, miR-218, miR-220, miR-221, miR-222, miR-223, miR-224, miR-296, miR-299, miR-301, miR-302a*, miR-302a, miR-302b*, miR-302b, miR-302d, miR-302c*, miR-302c, miR-320, miR-323, miR-324-3p, miR-324-5p, miR-325, miR-326, miR-328, miR-330, miR-331, miR-337, miR-338, miR-339, miR-340, miR-342, miR-345, miR-346, miR-363, miR-367, miR-368, miR-370, miR-371, miR-372, miR-373*, miR-373, miR-374, miR-375, miR-376b, miR-378, miR-379, miR-380-5p, miR-380-3p, miR-381, miR-382, miR-383, miR-410, miR-412, miR-422a, miR-422b, miR-423, miR-424, miR-425, miR-429, miR-431, miR-448, miR-449, miR-450, miR-451, let7a, let7b, let7c, let7d, let7e, let7f, let7g, let7i, miR-376a, and miR-377. The sequences of the above-mentioned microRNA targets are provided in “the miRBase sequence database” as described in Griffith-Jones et al. (2004), Nucleic Acids Research 32:D109-D111, and Griffith-Jones et al. (2006), Nucleic Acids Research 34:D140-D144, which is publicly accessible on the World Wide Web at the Wellcome Trust Sanger Institute website at http://microrna.sanger.ac.uk/sequences/.

Exemplary primers for use in accordance with this embodiment of the kit are provided in TABLE 1, TABLE 2, and TABLE 6 below.

In another embodiment, the kit comprises one or more primer sets capable of detecting at least one or more of the following human microRNA target templates: miR-1, miR-7, miR-10b, miR-26a, miR-26b, miR-29a, miR-30e-3p, miR-95, miR-107, miR-141, miR-143, miR-154*, miR-154, miR-155, miR-181a, miR-181b, miR-181c, miR-190, miR-193, miR-194, miR-195, miR-202, miR-206, miR-208, miR-212, miR-221, miR-222, miR-224, miR-296, miR-299, miR-302c*, miR-302c, miR-320, miR-339, miR363, miR-376b, miR379, miR410, miR412, miR424, miR429, miR431, miR449, miR451, let7a, let7b, let7c, let7d, let7e, let7f, let7g, and let7i. Exemplary primers for use in accordance with this embodiment of the kit are provided in TABLE 1, TABLE 2, and TABLE 6 below.

In another embodiment, the kit comprises one or more primer sets capable of detecting at least one or more of the following human, mouse or rat microRNA target templates: miR-1, miR-9, miR-18b, miR-20b, miR-92b, miR-146b, miR-181d, miR-193b, miR-194, miR-206, miR-291a-3p, miR-291b-3p, miR-301b, miR-329, miR-346, miR-351, miR-362, miR-362-3p, miR-369-5p, miR-384, miR-409-3p, miR-409-5p, miR-425-5p, miR-449b, miR-455, miR-483, miR-484, miR-485-3p, miR-485-5p, miR-486, miR-487b, miR-488, miR-489, miR-490, miR-491, miR-493-3p, miR-494, miR-495, miR-497, miR-499, miR-500, miR-501, miR-503, miR-505, miR-519a, miR-519b, miR-519c, miR-519d, miR-520a, miR-520b, miR-520d, miR-520e, miR-520f, miR-532, miR-539, miR-542-3p, miR-542-5p, miR-615, miR-652, miR-668, miR-671, miR-675-5p, miR-699, miR-721, and miR-758.

Exemplary primers for use in accordance with this embodiment of the kit are provided in TABLE 8.

In another embodiment, the kit comprises at least one oligonucleotide primer selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 493, as shown in TABLE 1, TABLE 2, TABLE 6, and TABLE 7.

In another embodiment, the kit comprises at least one oligonucleotide primer selected from the group consisting of SEQ ID NO: 47, 48, 49, 50, 55, 56, 81, 82, 83, 84, 91, 92, 103, 104, 123, 124, 145, 146, 193, 194, 197, 198, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 239, 240, 247, 248, 253, 254, 255, 256, 257, 258, 277, 278, 285, 286, 287, 288, 293, 294, 301, 302, 309, 310, 311, 312, 315, 316, 317, 318, 319, 320, 333, 334, 335, 336, 337, 338, 359, 360, 369, 370, 389, 390, 393, 394, 405, 406, 407, 408, 415, 416, 419, 420, 421, 422, 425, 426, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 461 and 462, as shown in TABLE 6.

In another embodiment, the kit comprises at least one oligonucleotide primer selected from the group consisting of SEQ ID NO: 500 to SEQ ID NO: 965, as shown in TABLE 8.

A kit of the invention can also provide reagents for primer extension and amplification reactions. For example, in some embodiments, the kit may further include one or more of the following components: a reverse transcriptase enzyme, a DNA polymerase enzyme, a Tris buffer, a potassium salt (e.g., potassium chloride), a magnesium salt (e.g., magnesium chloride), a reducing agent (e.g., dithiothreitol), and deoxynucleoside triphosphates (dNTPs).

In various embodiments, the kit may include a detection reagent such as SYBR green dye or BEBO dye that preferentially or exclusively binds to double stranded DNA during a PCR amplification step. In other embodiments, the kit may include a forward and/or reverse primer that includes a fluorophore and quencher to measure the amount of the PCR amplification products.

The kit optionally includes instructions for using the kit in the detection and quantitation of one or more mammalian microRNA targets. The kit can also be optionally provided in a suitable housing that is preferably useful for robotic handling in a high throughput manner.

The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention.

Example 1

This Example describes a representative method of the invention for producing DNA molecules from microRNA target molecules.

Primer extension was conducted as follows (using InVitrogen SuperScript III® reverse transcriptase and following the guidelines that were provided with the enzyme). The following reaction mixture was prepared on ice:

    • 1 μl of 10 mM dNTPs
    • 1 μl of 21.1M extension primer
    • 1-5 μl of target template
    • 4 μl of “5×cDNA buffer”
    • 1 μl of 0.1 M DTT
    • 1 μl of RNAse OUT
    • 1 μl of SuperScript III® enzyme
    • water to 20

The mixture was incubated at 50° C. for 30 minutes, then 85° C. for 5 minutes, then cooled to room temperature and diluted 10-fold with TE (10 mM Tris, pH 7.6, 0.1 mM EDTA).

Real-time PCR was conducted using an ABI 7900 HTS detection system (Applied Biosystems, Foster City, Calif., U.S.A.) by monitoring SYBR® green fluorescence of double-stranded PCR amplicons as a function of PCR cycle number. A typical 101.11 PCR reaction mixture contained:

    • 5 μl of 2×SYBR® green master mix (ABI)
    • 0.8 μl of 10 μM universal forward primer
    • 0.8 μl of 10 μM reverse primer
    • 1.4 μl of water
    • 2.0 μl of target template (10-fold diluted RT reaction)

The reaction was monitored through 40 cycles of standard “two cycle” PCR (95° C.-15 sec, 60° C.-60 sec) and the fluorescence of the PCR products was measured.

The foregoing method was successfully used in eleven primer extension PCR assays for quantitation of endogenous microRNAs present in a sample of total RNA. The DNA sequences of the extension primers, the universal forward primer sequence, and the LNA substituted reverse primers, used in these 11 assays are shown in TABLE 1.

TABLE 1 Target Primer Primer SEQ microRNA number Name DNA sequence (5′ to 3′) ID NO gene-specific extension primers1 humanb let7a 357 let7aP4 CATGATCAGCTGGGCCAAGAAACTATACAACCT 2 human miR-1 337 miR1P5 CATGATCAGCTGGGCCAAGATACATACTTCT 3 human miR-15a 344 miR15aP3 CATGATCAGCTGGGCCAAGACACAAACCATTATG 4 human miR-16 351 miR16P2 CATGATCAGCTGGGCCAAGACGCCAATATTTACGT 5 human miR-21 342 miR21P6 CATGATCAGCTGGGCCAAGATCAACATCAGT 6 human miR-24 350 miR24P5 CATGATCAGCTGGGCCAAGACTGTTCCTGCTG 7 human miR-122 222 122-E5F CATGATCAGCTGGGCCAAGAACAAACACCATTGTCA 8 human miR-124 226 124-E5F CATGATCAGCTGGGCCAAGATGGCATTCACCGCGTG 9 human miR-143 362 miR143P5 CATGATCAGCTGGGCCAAGATGAGCTACAGTG 10 human miR-145 305 miR145P2 CATGATCAGCTGGGCCAAGAAAGGGATTCCTGGGAA 11 human miR-155 367 miR155P3 CATGATCAGCTGGGCCAAGACCCCTATCACGAT 12 universal forward primer 230 E5F CATGATCAGCTGGGCCAAGA 13 RNA species-specific reverse primers2 human let7a 290 miRlet7a- TG+AGGT+AGTAGGTTG 14 1,2,3R human miR-1 285 miR1-1,2R TG+GAA+TG+TAAAGAAGTA 15 human miR-15a 287 miR15aR TAG+CAG+CACATAATG 16 human miR-16 289 miR16-1,2R T+AGC+AGCACGTAAA 17 human miR-21 286 miR21R T+AG+CT+TATCAGACTGAT 18 human miR-24 288 miR24-1,2R TGG+CTCAGTTCAGC 19 human miR-122 234 122LNAR T+G+GAG+TGTGACAA 20 human miR-124 235 124LNAR T+TAA+GGCACGCG 21 human miR-143 291 miR143R TG+AGA+TGAAGCACTG 22 human miR-145 314 miR145R2 GT+CCAGTTTTCCCA 23 human miR-155 293 miR155R T+TAA+TG+CTAATCGTGA 24 1Universal forward primer binding sites are shown in italics. The overlap with the RNA-specific reverse primers are underlined. 2LNA molecules are preceded by a “+”. Region of overlap of the reverse primers with the corresponding extension primers are underlined.

The assay was capable of detecting microRNA in a concentration range of from 2 nM to 20 fM. The assays were linear at least up to a concentration of 2 nM of synthetic microRNA (>1,000,000 copies/cell).

Example 2

This Example describes the evaluation of the minimum sequence requirements for efficient primer-extension mediated cDNA synthesis using a series of extension primers for microRNA assays having gene specific regions that range in length from 12 to 3 base pairs.

Primer Extension Reactions. Primer extension was conducted using the target molecules miR-195 and miR-215 as follows. The target templates miR-195 and miR-215 were diluted to 1 nM RNA (100,000 copies/cell) in TE zero plus 100 ng/μl total yeast RNA. A no template control (NTC) was prepared with TE zero plus 100 ng/μl total yeast RNA.

The reverse transcriptase reactions were carried out as follows (using InVitrogen SuperScript III® reverse transcriptase and following the guidelines that were provided with the enzyme) using a series of extension primers for miR-195 (SEQ ID NO: 25-34) and a series of extension primers for miR-215 (SEQ ID NO: 35-44) the sequences of which are shown below in TABLE 2.

The following reaction mixtures were prepared on ice:

    • Set 1: No Template Control
    • 37.5 μl water
    • 12.5 μl of 10 mM dNTPs
    • 12.5 μl 0.1 mM DTT
    • 50 μl of “5×cDNA buffer”
    • 12.5 μl RNAse OUT
    • 12.5 μl Superscript III® reverse transcriptase enzyme
    • 12.5 μl 1 μg/μl Hela cell total RNA (Ambion)
    • plus 50 μl of 2 μM extension primer
    • plus 50 μl TEzero+yeast RNA
    • Set 2: Spike-in Template
    • 37.5 μl water
    • 12.5 μl of 10 mM dNTPs
    • 12.5 μl 0.1 mM DTT
    • 50 μl of “5×cDNA buffer”
    • 12.5 μl RNAse OUT
    • 12.5 μl Superscript III® reverse transcriptase enzyme (InVitrogen)
    • 12.5 μl 1 μg/μl Hela cell total RNA (Ambion)
    • plus 50 μl of 2 μM extension primer
    • plus 50 μl 1 nM RNA target template (miR-195 or miR-215)
    • serially diluted in 10-fold increments

The reactions were incubated at 50° C. for 30 minutes, then 85° C. for 5 minutes, and cooled to 4° C. and diluted 10-fold with TE (10 mM Tris, pH 7.6, 0.1 mM EDTA).

Quantitative Real-Time PCR Reactions. Following reverse transcription, quadruplicate measurements of cDNA were made by quantitative real-time (qPCR) using an ABI 7900 HTS detection system (Applied Biosystems, Foster City, Calif., U.S.A.) by monitoring SYBR® green fluorescence of double-stranded PCR amplicons as a function of PCR cycle number. The following reaction mixture was prepared:

    • 5 μl of 2×SYBR green master mix (ABI)
    • 0.8 μl of 10 μM universal forward primer (SEQ ID NO: 13)
    • 0.8 μl of 10 μM reverse primer (miR-195RP:SEQ ID NO: 45 or
    • miR215RP: SEQ ID NO: 46)
    • 1.4 μl of water
    • 2.0 μl of target template (10-fold diluted miR-195 or miR-215 RT reaction)

Quantitative real-time PCR was performed for each sample in quadruplicate, using the manufacturer's recommended conditions. The reactions were monitored through 40 cycles of standard “two cycle” PCR (95° C.-15 sec, 60° C.-60 sec) and the fluorescence of the PCR products were measured and disassociation curves were generated. The DNA sequences of the extension primers, the universal forward primer sequence, and the LNA substituted reverse primers, used in the miR-195 and miR-215 assays are shown below in TABLE 2. The assay results for miR-195 are shown below in TABLE 3 and the assay results for miR-215 are shown below in TABLE 4.

TABLE 2 Target Primer SEQ ID microRNA number Primer Name DNA sequence (5′ to 3′) NO: gene-specific extension primers1 miR-195 646 mir195-GS1 CATGATCAGCTGGGCCAAGAGCCAATATTTCT 25 miR-195 647 mir195-GS2 CATGATCAGCTGGGCCAAGAGCCAATATTTC 26 miR-195 648 mir195-GS3 CATGATCAGCTGGGCCAAGAGCCAATATTT 27 miR-195 649 mir195-GS4 CATGATCAGCTGGGCCAAGAGCCAATATT 28 miR-195 650 mir195-GS5 CATGATCAGCTGGGCCAAGAGCCAATAT 29 miR-195 651 mir195-GS6 CATGATCAGCTGGGCCAAGAGCCAATA 30 miR-195 652 mir195-GS7 CATGATCAGCTGGGCCAAGAGCCAAT 31 miR-195 653 mir195-GS8 CATGATCAGCTGGGCCAAGAGCCAA 32 miR-195 654 mir195-GS9 CATGATCAGCTGGGCCAAGAGCCA 33 miR-195 655 mir195-GS10 CATGATCAGCTGGGCCAAGAGCC 34 miR-215 656 mir215-GS1 CATGATCAGCTGGGCCAAGAGTCTGTCAATTC 35 miR-215 657 mir215-GS2 CATGATCAGCTGGGCCAAGAGTCTGTCAATT 36 miR-215 658 mir215-GS3 CATGATCAGCTGGGCCAAGAGTCTGTCAAT 37 miR-215 659 mir215-GS4 CATGATCAGCTGGGCCAAGAGTCTGTCAA 38 miR-215 660 mir215-GS5 CATGATCAGCTGGGCCAAGAGTCTGTCA 39 miR-215 661 mir215-GS6 CATGATCAGCTGGGCCAAGAGTCTGTC 40 miR-215 662 mir215-GS7 CATGATCAGCTGGGCCAAGAGTCTGT 41 miR-215 663 mir215-GS8 CATGATCAGCTGGGCCAAGAGTCTG 42 miR-215 664 mir215-GS9 CATGATCAGCTGGGCCAAGAGTCT 43 miR-215 665 mir215-GS10 CATGATCAGCTGGGCCAAGAGTC 44 RNA species-specific reverse primers2 miR-195 442 mir195RP T+AGC+AGCACAGAAAT 45 miR-215 446 mir215RP A+T+GA+CCTATGAATTG 46 1Universal forward primer binding sites are shown in italics. 2The “+” symbol precedes the LNA molecules.

Results:

The sensitivity of each assay was measured by the cycle threshold (Ct) value which is defined as the cycle count at which fluorescence was detected in an assay containing microRNA target template. The lower this Ct value (e.g. the fewer number of cycles), the more sensitive was the assay. For microRNA samples, it was generally observed that while samples that contain template and no template controls both eventually cross the detection threshold, the samples with template do so at a much lower cycle number. The ΔCt value is the difference between the number of cycles (Ct) between template containing samples and no template controls, and serves as a measure of the dynamic range of the assay. Assays with a high dynamic range allow measurements of very low microRNA copy numbers. Accordingly, desirable characteristics of a microRNA detection assay include high sensitivity (low Ct value) and broad dynamic range (ΔCt≧12) between the signal of a sample containing target template and a no template background control sample.

The results of the miR195 and miR215 assays using extension primers having a gene specific portion ranging in size from 12 nucleotides to 3 nucleotides are shown below in TABLE 3 and TABLE 4, respectively. The results of these experiments unexpectedly demonstrate that gene-specific priming sequences as short as 3 nucleotides exhibit template specific priming. For both the miR-195 assay sets (shown in TABLE 3) and the miR-215 assay sets (shown in TABLE 4), the results demonstrate that the dynamic range (ΔCt) for both sets of assays are fairly consistent for extension primers having gene specific regions that are greater or equal to 8 nucleotides in length. The dynamic range of the assay (ΔCt) begins to decrease for extension primers having gene specific regions below 8 nucleotides, with a reduction in assay specificity below 7 nucleotides in the miR-195 assays, and below 6 nucleotides in the miR-215 assays. A melting point analysis of the miR-215 samples demonstrated that even at 3 nucleotides, there is specific PCR product present in the plus template samples (data not shown). Taken together, these data demonstrate that the gene specific region of extension primers is ideally ≧8 nucleotides, but can be as short as 3 nucleotides in length.

TABLE 3 MIR195 ASSAY RESULTS GS Primer Ct: No Ct: Plus Length Template Control Template Δ Ct 12 34.83 20.00 14.82 12 34.19 19.9 14.3 11 40.0 19.8 20.2 10 36.45 21.2 15.2 9 36.40 22.2 14.2 8 40.0 23.73 16.27 7 36.70 25.96 10.73 6 30.95 26.58 4.37 5 30.98 31.71 −0.732 4 32.92 33.28 −0.364 3 35.98 35.38 −0.605 Ct = the cycle count where the fluorescence exceeds the threshold of detection. Δ Ct = the difference between the Ct value with template and no template.

TABLE 4 MIR215 ASSAY RESULTS GS Primer Ct: No Ct: Plus Length Template Control Template Δ Ct 12 33.4 13.57 19.83 12 33.93 14.15 19.77 11 35.51 15.76 19.75 10 35.33 15.49 19.84 9 36.02 16.84 19.18 8 35.79 17.07 18.72 7 32.29 17.58 14.71 6 34.38 20.62 13.75 5 34.41 28.65 5.75 4 36.36 33.92 2.44 3 35.09 33.38 1.70 Ct = the cycle count where the fluorescence exceeds the threshold of detection. Δ Ct = the difference between the Ct value with template and no template.

Example 3

This Example describes assays and primer sets designed for quantitative analysis of human microRNA expression patterns.

Primer Design:

microRNA target templates: the sequence of the target templates as described herein are publicly available accessible on the World Wide Web at the Wellcome Trust Sanger Institute Web site in the “miRBase sequence database” as described in Griffith-Jones et al. (2004), Nucleic Acids Research 32:D109-D111, and Griffith-Jones et al. (2006), Nucleic Acids Research 34:D140-D144.

Extension primers: gene specific primers for primer extension of a microRNA to form a cDNA followed by quantitative PCR (qPCR) amplification were designed to (1) convert the RNA template into cDNA; (2) to introduce a “universal” PCR binding site (SEQ ID NO:1) to one end of the cDNA molecule; and (3) to extend the length of the cDNA to facilitate subsequent monitoring by qPCR.

Reverse primers: unmodified reverse primers and locked nucleic acid (LNA) containing reverse primers (RP) were designed to quantify the primer-extended, full length cDNA in combination with a generic universal forward primer (SEQ ID NO:13). For the locked nucleic acid containing reverse primers, two or three LNA modified bases were substituted within the first 8 nucleotides from the 5′ end of the reverse primer oligonucleotide, as shown below in the exemplary reverse primer sequences provided in TABLE 6. The LNA base substitutions were selected to raise the predicted Tm of the primer by the highest amount, and the final predicted Tm of the selected primers were specified to be preferably less than or equal to 55° C.

An example describing an assay utilizing an exemplary set of primers the detection of miR-95 and miR-424 is described below.

Primer Extension Reactions: primer extension was conducted using DNA templates corresponding to miR-95 and miR-424 as follows. The DNA templates were diluted to 0 nM, 1 nM, 100 pM, 10 pM, and 1 pM dilutions in TE zero (10 mM Tris pH 7.6, 0.1 mM EDTA) plus 100 ng/μl yeast total RNA (Ambion, Austin, Tex.).

The reverse transcriptase reactions were carried out using the following primers:

Extension primers: (diluted to 500 nM)

miR-95GSP CATGATCAGCTGGGCCAAGATGCTCAATAA (SEQ ID NO: 123) miR-424GSP CATGATCAGCTGGGCCAAGATTCAAAACAT (SEQ ID NO: 415)

Reverse primers: (diluted to 10 mM)

miR-95_RP4 TT+CAAC+GGGTATTTATTGA (SEQ ID NO: 124) miR-424RP2 C+AG+CAGCAATTCATGTTTT (SEQ ID NO: 416)

Reverse Transcription (per reaction):

2 μl water

2 μl of “5×cDNA buffer” (InVitrogen, Carlsbad, Calif.)

0.5 μl of 0.1 mM DTT (InVitrogen, Carlsbad, Calif.)

0.5 μl of 10 mM dNTPs (InVitrogen, Carlsbad, Calif.)

0.5 μl RNAse OUT (InVitrogen, Carlsbad, Calif.)

0.5 μl Superscript III® reverse transcriptase enzyme (InVitrogen, Carlsbad, Calif.)

2 μl of extension primer plus 2 μl of template dilution

The reactions were mixed and incubated at 50° C. for 30 minutes, then 85° C. for 5 minutes, and cooled to 4° C. and diluted 10-fold with TE zero.

Quantitative Real-Time PCR Reactions (per reaction):

    • 5 μl 12×SYBR mix (Applied Biosystems, Foster City, Calif.)
    • 1.4 μl water
    • 0.8 μl universal primer (CATGATCAGCTGGGCCAAGA (SEQ ID NO: 13))
    • 2.0 μl of diluted reverse transcription (RT) product from above.

Quantitative real-time PCR was performed for each sample in quadruplicate, using the manufacturer's recommended conditions. The reactions were monitored through 40 cycles of standard “two cycle” PCR (95° C.-15 sec, 60° C.-60 sec) and the fluorescence of the PCR products were measured and disassociation curves were generated. The DNA sequences of the extension primers, the universal forward primer sequence, and the LNA substituted reverse primers, used in the representative miR-95 and miR-424 assays as well as primer sets for 212 different human microRNA templates are shown below in TABLE 6. Primer sets for assays requiring extensive testing and design modification to achieve a sensitive assay with a high dynamic range are indicated in TABLE 6 with the symbol # following the primer name.

Results:

TABLE 5 shows the Ct values (averaged from four samples) from the miR-95 and miR-424 assays, which are plotted in the graph shown in FIG. 2. The results of these assays are provided as representative examples in order to explain the significance of the assay parameters shown in TABLE 6 designated as slope (column 6), intercept (column 7) and background (column 8).

As shown in TABLE 5, the Ct value for each template at various concentrations is provided. The Ct values (x-axis) are plotted as a function of template concentration (y-axis) to generate a standard curve for each assay, as shown in FIG. 2. The slope and intercept define the assay measurement characteristics that permit an estimation of number of copies/cell for each microRNA. For example, when the Ct values for 50 μg total RNA input for the miR-95 assay are plotted, a standard curve is generated with a slope and intercept of −0.03569 and 9.655, respectively. When these standard curve parameters are applied to the Ct of an unknown sample (x), they yield log 10 (copies/20 pg total RNA) (y). Because the average cell yields 20 pg of total RNA, these measurements equate to copies of microRNA/cell. The background provides an estimate of the minimum copy number that can be measured in a sample and is computed by inserting the no template control (NTC) value into this equation. In this example, as shown in TABLE 6, miR-95 yields a background of 1.68 copies/20 pg at 50 μg of RNA input.

As further shown in TABLE 6, reverse primers that do not contain LNA may also be used in accordance with the methods of the invention. See, e.g., SEQ ID NO:494-499. The sensitivity and dynamic range of the assays using non-LNA containing reverse primers SEQ ID NO:494-499, yielded similar results to the corresponding assays using LNA-containing reverse primers.

TABLE 5 Ct Values (averaged from four samples) Template concentration 10 nM 1 nM 0.1 nM 0.01 nM 0.001 nM NTC copies/20 pg  500,000  50,000  5000  500  50 RNA (50 μg input) copies/20 pg 5,000,000 500,000 50,000 5000 500 RNA (5 μg input) miR-95 11.71572163 14.17978 17.46353 19.97259 23.33171 27.44383 miR-424 10.47708975 12.76806 15.69251 18.53729 21.56897 23.2813  log10 (copies  5.698970004  4.69897  3.69897  2.69897  1.69897 for 50 μg input)

TABLE 6 PRIMERS TO DETECT HUMAN MICRORNA TARGET TEMPLATES Human Target Background micro RNA input RNA Extension Primer Name Extension Primer Sequence Reverse Primer Name Reverse Primer Sequence Slope Intercept 50 ug 5 ug miR-1 miR1GSP10# CATGATCAGCTGGGCCAAGATACATACTTC miR-1RP# T+G+GAA+TG+TAAAGAAGT −0.2758 8.3225 2.44 24.36 SEQ ID NO: 47 SEQ ID NO: 48 miR-7 miR-7GSP10# CATGATCAGCTGGGCCAAGACAACAAAATC miR-7_RP6# T+GGAA+GACTAGTGATTTT −0.2982 10.435 11.70 116.99 SEQ ID NO: 49 SEQ ID NO: 50 miR-9* miR-9*GSP CATGATCAGCTGGGCCAAGAACTTTCGGTT miR-9*RP TAAA+GCT+AGATAACCG −0.2405 8.9145 3.71 37.15 SEQ ID NO: 51 SEQ ID NO: 52 miR-10a miR-10aGSP CATGATCAGCTGGGCCAAGACACAAATTCG miR-10aRP T+AC+CCTGTAGATCCG −0.2755 8.6976 0.09 0.94 SEQ ID NO: 53 SEQ ID NO: 54 miR-10b miR- CATGATCAGCTGGGCCAAGAACAAATTCGGT miR- TA+CCC+TGT+AGAACCGA −0.3505 8.7109 0.55 5.52 10b_GSP11# SEQ ID NO: 55 10b_RP2# SEQ ID NO: 56 miR-15a miR-15aGSP CATGATCAGCTGGGCCAAGACACAAACCAT miR-15aRP T+AG+CAGCACATAATG −0.2831 8.4519 4.40 44.01 SEQ ID NO: 57 SEQ ID NO: 58 miR-15b miR-15bGSP2 CATGATCAGCTGGGCCAAGATGTAAACCA miR-15bRP T+AG+CAGCACATCAT −0.2903 8.4206 0.18 1.84 SEQ ID NO: 59 SEQ ID NO: 60 miR-16 miR-16GSP2 CATGATCAGCTGGGCCAAGACGCCAATAT miR-16RP T+AG+CAGCACGTAAA −0.2542 9.3689 1.64 16.42 SEQ ID NO: 61 SEQ ID NO: 62 miR-17- miR-17-3pGSP CATGATCAGCTGGGCCAAGAACAAGTGCCT miR-17-3pRP A+CT+GCAGTGAAGGC −0.2972 8.2625 1.08 10.78 3p SEQ ID NO: 63 SEQ ID NO: 64 miR-17- miR-17- CATGATCAGCTGGGCCAAGAACTACCTGC miR-17-5pRP C+AA+AGTGCTTACAGTG −0.2956 7.9101 0.13 1.32 5p 5pGSP2 SEQ ID NO: 65 SEQ ID NO: 66 miR-19a miR-19aGSP2 CATGATCAGCTGGGCCAAGATCAGTTTTG miR-19aRP TG+TG+CAAATCTATGC −0.2984 9.461 0.02 0.23 SEQ ID NO: 67 SEQ ID NO: 68 miR-19b miR-19bGSP CATGATCAGCTGGGCCAAGATCAGTTTTGC miR-19bRP TG+TG+CAAATCCATG −0.294 8.1434 2.26 22.55 SEQ ID NO: 69 SEQ ID NO: 70 miR-20 miR-20GSP3 CATGATCAGCTGGGCCAAGACTACCTGC miR-20RP T+AA+AGTGCTTATAGTGCA −0.2979 7.9929 0.16 1.60 SEQ ID NO: 71 SEQ ID NO: 72 miR-21 miR-21GSP2 CATGATCAGCTGGGCCAAGATCAACATCA miR-21RP T+AG+CTTATCAGACTGATG −0.2849 8.1624 1.80 17.99 SEQ ID NO: 73 SEQ ID NO: 74 miR-23a miR-23aGSP CATGATCAGCTGGGCCAAGAGGAAATCCCT miR-23aRP A+TC+ACATTGCCAGG −0.3172 9.4253 2.41 24.08 SEQ ID NO: 75 SEQ ID NO: 76 miR-23b miR-23bGSP CATGATCAGCTGGGCCAAGAGGTAATCCCT miR-23bRP A+TC+ACATTGCCAGG −0.2944 9.0985 5.39 53.85 SEQ ID NO: 77 SEQ ID NO: 78 miR-25 miR-25GSP CATGATCAGCTGGGCCAAGATCAGACCGAG miR-25RP C+AT+TGCACTTGTCTC −0.3009 8.2482 1.52 15.19 SEQ ID NO: 79 SEQ ID NO: 80 miR-26a miR-26aGSP9# CATGATCAGCTGGGCCAAGAGCCTATCCT miR- TT+CA+AGTAATCCAGGAT −0.2807 8.558 0.26 2.56 SEQ ID NO: 81 26aRP2# SEQ ID NO: 82 miR-26b miR-26bGSP9# CATGATCAGCTGGGCCAAGAAACCTATCC miR- TT+CA+AGT+AATTCAGGAT −0.2831 8.7885 0.37 3.67 SEQ ID NO: 83 26bRP2# SEQ ID NO: 84 miR-27a miR-27aGSP CATGATCAGCTGGGCCAAGAGCGGAACTTA miR-27aRP TT+CA+CAGTGGCTAA −0.2765 9.5239 5.15 51.51 SEQ ID NO: 85 SEQ ID NO: 86 miR-27b miR-27bGSP CATGATCAGCTGGGCCAAGAGCAGAACTTA miR-27bRP TT+CA+CAGTGGCTAA −0.28 9.5483 5.97 59.71 SEQ ID NO: 87 SEQ ID NO: 88 miR-28 miR-28GSP CATGATCAGCTGGGCCAAGACTCAATAGAC miR-28RP A+AG+GAGCTCACAGT −0.3226 10.071 7.19 71.87 SEQ ID NO: 89 SEQ ID NO: 90 miR-29a miR-29aGSP8# CATGATCAGCTGGGCCAAGAAACCGATT miR- T+AG+CACCATCTGAAAT −0.29 8.8731 0.04 0.38 SEQ ID NO: 91 29aRP2# SEQ ID NO: 92 miR-29b miR-29bGSP2 CATGATCAGCTGGGCCAAGAAACACTGAT miR-29bRP2 T+AG+CACCATTTGAAATCAG −0.3162 9.6276 3.56 35.57 SEQ ID NO: 93 SEQ ID NO: 94 miR-30a- miR-30a- CATGATCAGCTGGGCCAAGACTTCCAGTCG miR-30a- T+GT+AAACATCCTCGAC −0.2772 9.0694 1.92 19.16 5p 5pGSP SEQ ID NO: 95 5pRP SEQ ID NO: 96 miR-30b miR-30bGSP CATGATCAGCTGGGCCAAGAAGCTGAGTGT miR-30bRP TGT+AAA+CATCCTACACT −0.2621 8.5974 0.11 1.13 SEQ ID NO: 97 SEQ ID NO: 98 miR-30c miR-30cGSP CATGATCAGCTGGGCCAAGAGCTGAGAGTG miR-30cRP TGT+AAA+CATCCTACACT −0.2703 8.699 0.15 1.48 SEQ ID NO: 99 SEQ ID NO: 100 miR-30d miR-30dGSP CATGATCAGCTGGGCCAAGACTTCCAGTCG miR-30dRP T+GTAAA+CATCCCCG −0.2506 9.3875 0.23 2.31 SEQ ID NO: 101 SEQ ID NO: 102 miR-30e- miR-30e- CATGATCAGCTGGGCCAAGAGCTGTAAAC miR-30e- CTTT+CAGT+CGGATGTTT −0.325 11.144 6.37 63.70 3p 3pGSP9# SEQ ID NO: 103 3pRP5# SEQ ID NO: 104 miR-30e- miR-30e- CATGATCAGCTGGGCCAAGATCCAGTCAAG miR-30e- TG+TAAA+CATCCTTGAC −0.2732 8.1604 8.50 85.03 5p 5pGSP SEQ ID NO: 105 5pRP SEQ ID NO: 106 miR-31 miR-31GSP CATGATCAGCTGGGCCAAGACAGCTATGCC miR-31RP G+GC+AAGATGCTGGC −0.3068 8.2605 3.74 37.43 SEQ ID NO: 107 SEQ ID NO: 108 miR-32 miR-32GSP CATGATCAGCTGGGCCAAGAGCAACTTAGT miR-32RP TATTG+CA+CATTACTAAG −0.2785 8.9581 0.39 3.93 SEQ ID NO: 109 SEQ ID NO: 110 miR-33 miR-33GSP2 CATGATCAGCTGGGCCAAGACAATGCAAC miR-33RP G+TG+CATTGTAGTTGC −0.3031 8.42 2.81 28.14 SEQ ID NO: 111 SEQ ID NO: 112 miR-34a miR-34aGSP CATGATCAGCTGGGCCAAGAAACAACCAGC miR-34aRP T+GG+CAGTGTCTTAG −0.3062 9.1522 2.40 23.99 SEQ ID NO: 113 SEQ ID NO: 114 miR-34b miR-34bGSP CATGATCAGCTGGGCCAAGACAATCAGCTA miR-34bRP TA+GG+CAGTGTCATT −0.3208 9.054 0.04 0.37 SEQ ID NO: 115 SEQ ID NO: 116 miR-34c miR-34cGSP CATGATCAGCTGGGCCAAGAGCAATCAGCT miR-34cRP A+GG+CAGTGTAGTTA −0.2995 10.14 1.08 10.83 SEQ ID NO: 117 SEQ ID NO: 118 miR-92 miR-92GSP CATGATCAGCTGGGCCAAGACAGGCCGGGA miR-92RP T+AT+TGCACTTGTCCC −0.3012 8.6908 8.92 89.17 SEQ ID NO: 119 SEQ ID NO: 120 miR-93 miR-93GSP CATGATCAGCTGGGCCAAGACTACCTGCAC miR-93RP AA+AG+TGCTGTTCGT −0.3025 7.9933 4.63 46.30 SEQ ID NO: 121 SEQ ID NO: 122 miR-95 miR-95GSP# CATGATCAGCTGGGCCAAGATGCTCAATAA miR- TT+CAAC+GGGTATTTATTGA −0.3436 9.655 1.68 16.80 SEQ ID NO: 123 95_RP4# SEQ ID NO: 124 miR-96 miR-96GSP CATGATCAGCTGGGCCAAGAGCAAAAATGT miR-96RP T+TT+GGCACTAGCAC −0.2968 9.2611 0.00 0.05 SEQ ID NO: 125 SEQ ID NO: 126 miR-98 miR-98GSP CATGATCAGCTGGGCCAAGAAACAATACAA miR-98RP TGA+GGT+AGTAAGTTG −0.2797 9.5654 1.05 10.48 SEQ ID NO: 127 SEQ ID NO: 128 miR-99a miR-99aGSP CATGATCAGCTGGGCCAAGACACAAGATCG miR-99aRP A+AC+CCGTAGATCCG −0.2768 8.781 0.21 2.08 SEQ ID NO: 129 SEQ ID NO: 130 miR-99b miR-99bGSP CATGATCAGCTGGGCCAAGACGCAAGGTCG miR-99bRP C+AC+CCGTAGAACCG −0.2747 7.9855 0.25 2.53 SEQ ID NO: 131 SEQ ID NO: 132 miR-100 miR-100GSP CATGATCAGCTGGGCCAAGACACAAGTTCG miR-100RP A+AC+CCGTAGATCCG −0.2902 8.669 0.04 0.35 SEQ ID NO: 133 SEQ ID NO: 134 miR-101 miR-101GSP CATGATCAGCTGGGCCAAGACTTCAGTTAT miR-101RP TA+CAG+TACTGTGATAACT −0.3023 8.2976 0.46 4.63 SEQ ID NO: 135 SEQ ID NO: 136 miR-103 miR-103GSP CATGATCAGCTGGGCCAAGATCATAGCCCT miR-103RP A+GC+AGCATTGTACA −0.3107 8.5776 0.02 0.21 SEQ ID NO: 137 SEQ ID NO: 138 miR-105 miR-105GSP CATGATCAGCTGGGCCAAGAACAGGAGTCT miR-105RP T+CAAA+TGCTCAGACT −0.2667 8.9832 0.93 9.28 SEQ ID NO: 139 SEQ ID NO: 140 miR-106a miR-106aGSP CATGATCAGCTGGGCCAAGAGCTACCTGCA miR-106aRP AAA+AG+TGCTTACAGTG −0.3107 8.358 0.03 0.31 SEQ ID NO: 141 SEQ ID NO: 142 miR-106b miR-106bGSP CATGATCAGCTGGGCCAAGAATCTGCACTG miR-106bRP T+AAAG+TGCTGACAGT −0.2978 8.7838 0.10 1.04 SEQ ID NO: 143 SEQ ID NO: 144 miR-107 miR-107GSP8# CATGATCAGCTGGGCCAAGATGATAGCC miR- A+GC+AGCATTGTACAG −0.304 9.1666 0.34 3.41 SEQ ID NO: 145 107RP2# SEQ ID NO: 146 miR-122a miR-122aGSP CATGATCAGCTGGGCCAAGAACAAACACCA miR-122aRP T+GG+AGTGTGACAAT −0.3016 8.1479 0.06 0.58 SEQ ID NO: 147 SEQ ID NO: 148 miR-124a miR-124aGSP CATGATCAGCTGGGCCAAGATGGCATTCAC miR-124aRP T+TA+AGGCACGCGGT −0.3013 8.6906 0.56 5.63 SEQ ID NO: 149 SEQ ID NO: 150 miR-125a miR-125aGSP CATGATCAGCTGGGCCAAGACACAGGTTAA miR-125aRP T+CC+CTGAGACCCTT −0.2938 8.6754 0.09 0.91 SEQ ID NO: 151 SEQ ID NO: 152 miR-125b miR-125bGSP CATGATCAGCTGGGCCAAGATCACAAGTTA miR-125bRP T+CC+CTGAGACCCTA −0.283 8.1251 0.20 1.99 SEQ ID NO: 153 SEQ ID NO: 154 miR-126 miR-126GSP CATGATCAGCTGGGCCAAGAGCATTATTAC miR-126RP T+CG+TACCGTGAGTA −0.26 8.937 0.18 1.80 SEQ ID NO: 155 SEQ ID NO: 156 miR-126* miR-126*GSP3 CATGATCAGCTGGGCCAAGACGCGTACC miR-126*RP C+ATT+ATTA+CTTTTGGTACG −0.2969 8.184 3.58 35.78 SEQ ID NO: 157 SEQ ID NO: 158 miR-127 miR-127GSP CATGATCAGCTGGGCCAAGAAGCCAAGCTC miR-127RP T+CG+GATCCGTCTGA −0.2432 9.1013 1.11 11.13 SEQ ID NO: 159 SEQ ID NO: 160 miR-128a miR-128aGSP CATGATCAGCTGGGCCAAGAAAAAGAGACC miR-128aRP T+CA+CAGTGAACCGG −0.2866 8.0867 0.16 1.60 SEQ ID NO: 161 SEQ ID NO: 162 miR-128b miR-128bGSP CATGATCAGCTGGGCCAAGAGAAAGAGACC miR-128bRP T+CA+CAGTGAACCGG −0.2923 8.0608 0.07 0.74 SEQ ID NO: 163 SEQ ID NO: 164 miR-129 miR-129GSP CATGATCAGCTGGGCCAAGAGCAAGCCCAG miR-129RP CTTTT+TG+CGGTCTG −0.2942 9.7731 0.88 8.85 SEQ ID NO: 165 SEQ ID NO: 166 miR-130a miR-130aGSP CATGATCAGCTGGGCCAAGAATGCCCTTTT miR-130aRP C+AG+TGCAATGTTAAAAG −0.2943 8.7465 1.28 12.78 SEQ ID NO: 167 SEQ ID NO: 168 miR-130b miR-130bGSP CATGATCAGCTGGGCCAAGAATGCCCTTTC miR-130bRP C+AG+TGCAATGATGA −0.2377 9.1403 3.14 31.44 SEQ ID NO: 169 SEQ ID NO: 170 miR-132 miR-132GSP CATGATCAGCTGGGCCAAGACGACCATGGC miR-132RP T+AA+CAGTCTACAGCC −0.2948 8.1167 0.11 1.13 SEQ ID NO: 171 SEQ ID NO: 172 miR-133a miR-133aGSP CATGATCAGCTGGGCCAAGAACAGCTGGTT miR-133aRP T+TG+GTCCCCTTCAA −0.295 9.3679 0.10 1.04 SEQ ID NO: 173 SEQ ID NO: 174 miR-133b miR-133bGSP CATGATCAGCTGGGCCAAGATAGCTGGTTG miR-133bRP T+TG+GTCCCCTTCAA −0.3062 8.3649 0.02 0.18 SEQ ID NO: 175 SEQ ID NO: 176 miR-134 miR-134GSP CATGATCAGCTGGGCCAAGACCCTCTGGTC miR-134RP T+GT+GACTGGTTGAC −0.2965 9.0483 0.14 1.39 SEQ ID NO: 177 SEQ ID NO: 178 miR-135a miR-135aGSP CATGATCAGCTGGGCCAAGATCACATAGGA miR-135aRP T+AT+GGCTTTTTATTCCT −0.2914 8.092 1.75 17.50 SEQ ID NO: 179 SEQ ID NO: 180 miR-135b miR-135bGSP CATGATCAGCTGGGCCAAGACACATAGGAA miR-135bRP T+AT+GGCTTTTCATTCC −0.2962 7.8986 0.05 0.49 SEQ ID NO: 181 SEQ ID NO: 182 miR-136 miR-136GSP CATGATCAGCTGGGCCAAGATCCATCATCA miR-136RP A+CT+CCATTTGTTTTGATG −0.3616 10.229 0.68 6.77 SEQ ID NO: 183 SEQ ID NO: 184 miR-137 miR-137GSP CATGATCAGCTGGGCCAAGACTACGCGTAT miR-137RP T+AT+TGCTTAAGAATACGC −0.2876 8.234 8.57 85.71 SEQ ID NO: 185 SEQ ID NO: 186 miR-138 miR-138GSP2 CATGATCAGCTGGGCCAAGACGGCCTGAT miR-138RP A+GC+TGGTGTTGTGA −0.3023 9.0814 0.22 2.19 SEQ ID NO: 187 SEQ ID NO: 188 miR-139 miR-139GSP CATGATCAGCTGGGCCAAGAAGACACGTGC miR-139RP T+CT+ACAGTGCACGT −0.2983 8.1141 6.92 69.21 SEQ ID NO: 189 SEQ ID NO: 190 miR-140 miR-140GSP CATGATCAGCTGGGCCAAGACTACCATAGG miR-140RP A+GT+GGTTTTACCCT −0.2312 8.3231 0.13 1.34 SEQ ID NO: 191 SEQ ID NO: 192 miR-141 miR-141GSP9# CATGATCAGCTGGGCCAAGACCATCTTTA miR- TAA+CAC+TGTCTGGTAA −0.2805 9.6671 0.13 1.26 SEQ ID NO: 193 141RP2# SEQ ID NO: 194 miR-142- miR-142- CATGATCAGCTGGGCCAAGATCCATAAA miR-142- TGT+AG+TGTTTCCTACT −0.2976 8.4046 0.03 0.27 3p 3pGSP3 SEQ ID NO: 195 3pRP SEQ ID NO: 196 miR-143 miR-143GSP8# CATGATCAGCTGGGCCAAGATGAGCTAC miR- T+GA+GATGAAGCACTG −0.3008 9.2675 0.37 3.71 SEQ ID NO: 197 143RP2# SEQ ID NO: 198 miR-144 miR-144GSP2 CATGATCAGCTGGGCCAAGACTAGTACAT miR-144RP TA+CA+GTAT+AGATGATG −0.2407 9.4441 0.95 9.52 SEQ ID NO: 199 SEQ ID NO: 200 miR-145 miR-145GSP2 CATGATCAGCTGGGCCAAGAAAGGGATTC miR-145RP G+TC+CAGTTTTCCCA −0.2937 8.0791 0.39 3.86 SEQ ID NO: 201 SEQ ID NO: 202 miR-146 miR-146GSP3 CATGATCAGCTGGGCCAAGAAACCCATG miR-146RP T+GA+GAACTGAATTCCA −0.2861 8.8246 0.08 0.75 SEQ ID NO: 203 SEQ ID NO: 204 miR-147 miR-147GSP CATGATCAGCTGGGCCAAGAGCAGAAGCAT miR-147RP G+TG+TGTGGAAATGC −0.2989 8.8866 1.65 16.47 SEQ ID NO: 205 SEQ ID NO: 206 miR-148a miR-148aGSP2 CATGATCAGCTGGGCCAAGAACAAAGTTC miR- T+CA+GTGCACTACAGAACT −0.2928 9.4654 1.27 12.65 SEQ ID NO: 207 148aRP2 SEQ ID NO: 208 miR-148b miR-148bGSP2 CATGATCAGCTGGGCCAAGAACAAAGTTC miR-148bRP T+CA+GTGCATCACAG −0.2982 10.417 0.24 2.44 SEQ ID NO: 209 SEQ ID NO: 210 miR-149 miR-149GSP2 CATGATCAGCTGGGCCAAGAGGAGTGAAG miR-149RP T+CT+GGCTCCGTGTC −0.2996 8.3392 2.15 21.50 SEQ ID NO: 211 SEQ ID NO: 212 miR-150 miR-150GSP3 CATGATCAGCTGGGCCAAGACACTGGTA miR-150RP T+CT+CCCAACCCTTG −0.2943 8.3945 0.06 0.56 SEQ ID NO: 213 SEQ ID NO: 214 miR-151 miR-151GSP2 CATGATCAGCTGGGCCAAGACCTCAAGGA miR-151RP A+CT+AGACTGAAGCTC −0.2975 8.651 0.16 1.60 SEQ ID NO: 215 SEQ ID NO: 216 miR-152 miR-152GSP2 CATGATCAGCTGGGCCAAGACCCAAGTTC miR-152RP T+CA+GTGCATGACAG −0.2741 8.7404 0.33 3.25 SEQ ID NO: 217 SEQ ID NO: 218 miR-153 miR-153GSP2 CATGATCAGCTGGGCCAAGATCACTTTTG miR-153RP TTG+CAT+AGTCACAAAA −0.2723 9.5732 3.32 33.19 SEQ ID NO: 219 SEQ ID NO: 220 miR-154* miR- CATGATCAGCTGGGCCAAGAAATAGGTCA miR- AATCA+TA+CACGGTTGAC −0.3056 8.8502 0.07 0.74 154*GSP9# SEQ ID NO: 221 154*RP2# SEQ ID NO: 222 miR-154 miR-154GSP9# CATGATCAGCTGGGCCAAGACGAAGGCAA miR- TA+GGTTA+TCCGTGTT −0.3062 9.3947 0.10 0.96 SEQ ID NO: 223 154RP3# SEQ ID NO: 224 miR-155 miR-155GSP8# CATGATCAGCTGGGCCAAGACCCCTATC miR- TT+AA+TGCTAATCGTGATAGG −0.3201 8.474 5.49 54.91 SEQ ID NO: 225 155RP2# SEQ ID NO: 226 miR-181a miR- CATGATCAGCTGGGCCAAGAACTCACCGA miR- AA+CATT+CAACGCTGTC −0.2919 7.968 1.70 17.05 181aGSP9# SEQ ID NO: 227 181aRP2# SEQ ID NO: 228 miR-181c miR- CATGATCAGCTGGGCCAAGAACTCACCGA miR- AA+CATT+CAACCTGTCG −0.3102 7.9029 1.08 10.78 181cGSP9# SEQ ID NO: 229 181cRP2# SEQ ID NO: 230 miR-182* miR-182*GSP CATGATCAGCTGGGCCAAGATAGTTGGCAA miR-182*RP T+GG+TTCTAGACTTGC −0.2978 8.5876 4.25 42.47 SEQ ID NO: 231 SEQ ID NO: 232 miR-182 miR-182GSP2 CATGATCAGCTGGGCCAAGATGTGAGTTC miR-182RP TTT+GG+CAATGGTAG −0.2863 9.0854 1.52 15.20 SEQ ID NO: 233 SEQ ID NO: 234 miR-183 miR-183GSP2 CATGATCAGCTGGGCCAAGACAGTGAATT miR-183RP T+AT+GGCACTGGTAG −0.2774 9.9254 1.95 19.51 SEQ ID NO: 235 SEQ ID NO: 236 miR-184 miR-184GSP2 CATGATCAGCTGGGCCAAGAACCCTTATC miR-184RP T+GG+ACGGAGAACTG −0.2906 7.9585 0.05 0.49 SEQ ID NO: 237 SEQ ID NO: 238 miR-186 miR-186GSP9# CATGATCAGCTGGGCCAAGAAAGCCCAAA miR- CA+AA+GAATT+CTCCTTTTGG −0.2861 8.6152 0.32 3.18 SEQ ID NO: 239 186RP3# SEQ ID NO: 240 miR-187 miR-187GSP CATGATCAGCTGGGCCAAGACGGCTGCAAC miR-187RP T+CG+TGTCTTGTGTT −0.2953 7.9329 1.23 12.31 SEQ ID NO: 241 SEQ ID NO: 242 miR-188 miR-188GSP CATGATCAGCTGGGCCAAGAACCCTCCACC miR-188RP C+AT+CCCTTGCATGG −0.2925 8.0782 8.49 84.92 SEQ ID NO: 243 SEQ ID NO: 244 miR-189 miR-189GSP2 CATGATCAGCTGGGCCAAGAACTGATATC miR-189RP G+TG+CCTACTGAGCT −0.2981 8.8964 0.21 2.08 SEQ ID NO: 245 SEQ ID NO: 246 miR-190 miR-190GSP9# CATGATCAGCTGGGCCAAGAACCTAATAT miR- T+GA+TA+TGTTTGATATATT −0.3317 9.8766 0.43 4.34 SEQ ID NO: 247 190RP4# AG SEQ ID NO: 248 miR-191 miR-191GSP2 CATGATCAGCTGGGCCAAGAAGCTGCTTT miR-191RP2 C+AA+CGGAATCCCAAAAG −0.299 9.0317 0.41 4.07 SEQ ID NO: 249 SEQ ID NO: 250 miR-192 miR-192GSP2 CATGATCAGCTGGGCCAAGAGGCTGTCAA miR-192RP C+TGA+CCTATGAATTGAC −0.2924 9.5012 1.10 10.98 SEQ ID NO: 251 SEQ ID NO: 252 miR-193 miR-193GSP9# CATGATCAGCTGGGCCAAGACTGGGACTT miR- AA+CT+GGCCTACAAAG −0.3183 8.9942 0.17 1.72 SEQ ID NO: 253 193RP2# SEQ ID NO: 254 miR-194 mir194GSP8# CATGATCAGCTGGGCCAAGATCCACATG mir194RP# TG+TAA+CAGCAACTCCA −0.3078 8.8045 0.37 3.69 SEQ ID NO: 255 SEQ ID NO: 256 miR-195 miR-195GSP9# CATGATCAGCTGGGCCAAGAGCCAATATT miR- T+AG+CAG+CACAGAAATA −0.2955 10.213 0.76 7.58 SEQ ID NO: 257 195RP3# SEQ ID NO: 258 miR-196b miR-196bGSP CATGATCAGCTGGGCCAAGACCAACAACAG miR-196bRP TA+GGT+AGTTTCCTGT −0.301 8.1641 1.47 14.66 SEQ ID NO: 259 SEQ ID NO: 260 miR-196a miR-196aGSP CATGATCAGCTGGGCCAAGACCAACAACAT miR-196aRP TA+GG+TAGTTTCATGTTG −0.2932 8.0448 8.04 80.37 SEQ ID NO: 261 SEQ ID NO: 262 miR-197 miR-197GSP2 CATGATCAGCTGGGCCAAGAGCTGGGTGG miR-197RP TT+CA+CCACCTTCTC −0.289 8.2822 0.71 7.10 SEQ ID NO: 263 SEQ ID NO: 264 miR-198 miR-198GSP3 CATGATCAGCTGGGCCAAGACCTATCTC miR-198RP G+GT+CCAGAGGGGAG −0.2986 8.1359 0.31 3.15 SEQ ID NO: 265 SEQ ID NO: 266 miR- miR- CATGATCAGCTGGGCCAAGAAACCAATGT miR- T+AC+AGTAGTCTGCAC −0.3029 9.0509 0.25 2.52 199a* 199a*GSP2 SEQ ID NO: 267 199a*RP SEQ ID NO: 268 miR-199a miR-199aGSP2 CATGATCAGCTGGGCCAAGAGAACAGGTA miR-199aRP C+CC+AGTGTTCAGAC −0.3187 9.2268 0.12 1.16 SEQ ID NO: 269 SEQ ID NO: 270 miR-199b miR-199bGSP CATGATCAGCTGGGCCAAGAGAACAGATAG miR-199bRP C+CC+AGTGTTTAGAC −0.3165 9.3935 2.00 20.04 SEQ ID NO: 271 SEQ ID NO: 272 miR-200a miR-200aGSP2 CATGATCAGCTGGGCCAAGAACATCGTTA miR-200aRP TAA+CAC+TGTCTGGT −0.2754 9.1227 0.08 0.78 SEQ ID NO: 273 SEQ ID NO: 274 miR-200b miR-200bGSP2 CATGATCAGCTGGGCCAAGAGTCATCATT miR-200bRP TAATA+CTG+CCTGGTAAT −0.2935 8.5461 0.08 0.85 SEQ ID NO: 275 SEQ ID NO: 276 miR-202 miR-202 CATGATCAGCTGGGCCAAGATTTTCCCATG miR-202RP# A+GA+GGTATA+GGGCAT −0.2684 9.056 0.25 2.48 GSP10# SEQ ID NO: 277 SEQ ID NO: 278 miR-203 miR-203GSP2 CATGATCAGCTGGGCCAAGACTAGTGGTC miR-203RP G+TG+AAATGTTTAGGACC −0.2852 8.1279 1.60 16.03 SEQ ID NO: 279 SEQ ID NO: 280 miR-204 miR-204GSP2 CATGATCAGCTGGGCCAAGAAGGCATAGG miR-204RP T+TC+CCTTTGTCATCC −0.2925 8.7648 0.16 1.59 SEQ ID NO: 281 SEQ ID NO: 282 miR-205 miR-205GSP CATGATCAGCTGGGCCAAGACAGACTCCGG miR-205RP T+CCTT+CATTCCACC −0.304 8.2407 9.21 92.15 SEQ ID NO: 283 SEQ ID NO: 284 miR-206 mir206GSP7# CATGATCAGCTGGGCCAAGACCACACA miR-206RP# T+G+GAA+TGTAAGGAAGTGT −0.2815 8.2206 0.29 2.86 SEQ ID NO: 285 SEQ ID NO: 286 miR-208 miR- CATGATCAGCTGGGCCAAGAACAAGCTTTTTGC miR- ATAA+GA+CG+AGCAAAAAG −0.2072 7.9097 57.75 577.52 208_GSP13# SEQ ID NO: 287 208_RP4# SEQ ID NO: 288 miR-210 miR-210GSP CATGATCAGCTGGGCCAAGATCAGCCGCTG miR-210RP C+TG+TGCGTGTGACA −0.2717 8.249 0.18 1.77 SEQ ID NO: 289 SEQ ID NO: 290 miR-211 miR-211GSP2 CATGATCAGCTGGGCCAAGAAGGCGAAGG miR-211RP T+TC+CCTTTGTCATCC −0.2926 8.3106 0.10 1.00 SEQ ID NO: 291 SEQ ID NO: 292 miR-212 miR-212GSP9# CATGATCAGCTGGGCCAAGAGGCCGTGAC miR- T+AA+CAGTCTCCAGTCA −0.2916 8.0745 0.59 5.86 SEQ ID NO: 293 212RP2# SEQ ID NO: 294 miR-213 miR-213GSP CATGATCAGCTGGGCCAAGAGGTACAATCA miR-213RP A+CC+ATCGACCGTTG −0.2934 8.1848 2.96 29.59 SEQ ID NO: 295 SEQ ID NO: 296 miR-214 miR-214GSP CATGATCAGCTGGGCCAAGACTGCCTGTCT miR-214RP A+CA+GCAGGCACAGA −0.2947 7.82 0.84 8.44 SEQ ID NO: 297 SEQ ID NO: 298 miR-215 miR-215GSP2 CATGATCAGCTGGGCCAAGAGTCTGTCAA miR-215RP A+TGA+CCTATGAATTGAC −0.2932 8.9273 1.51 15.05 SEQ ID NO: 299 SEQ ID NO: 300 miR-216 miR-216GSP9# CATGATCAGCTGGGCCAAGACACAGTTGC mir216RP# TAA+TCT+CAGCTGGCA −0.273 8.5829 0.95 9.50 SEQ ID NO: 301 SEQ ID NO: 302 miR-217 miR-217GSP2 CATGATCAGCTGGGCCAAGAATCCAATCA miR-217RP2 T+AC+TGCATCAGGAACTGA −0.3089 9.6502 0.07 0.71 SEQ ID NO: 303 SEQ ID NO: 304 miR-218 miR-218GSP2 CATGATCAGCTGGGCCAAGAACATGGTTA miR-218RP TTG+TGCTT+GATCTAAC −0.2778 8.4363 1.00 10.05 SEQ ID NO: 305 SEQ ID NO: 306 miR-220 miR-220GSP CATGATCAGCTGGGCCAAGAAAAGTGTCAG miR-220RP C+CA+CACCGTATCTG −0.2755 9.0728 8.88 88.75 SEQ ID NO: 307 SEQ ID NO: 308 miR-221 miR-221GSP9# CATGATCAGCTGGGCCAAGAGAAACCCAG miR-221RP# A+GC+TACATTGTCTGC −0.2886 8.5743 0.12 1.17 SEQ ID NO: 309 SEQ ID NO: 310 miR-222 miR-222GSP8# CATGATCAGCTGGGCCAAGAGAGACCCA miR-222RP# A+GC+TACATCTGGCT −0.283 8.91 1.64 16.41 SEQ ID NO: 311 SEQ ID NO: 312 miR-223 miR-223GSP CATGATCAGCTGGGCCAAGAGGGGTATTTG miR-223RP TG+TC+AGTTTGTCAAA −0.2998 8.6669 0.94 9.44 SEQ ID NO: 313 SEQ ID NO: 314 miR-224 miR-224GSP8# CATGATCAGCTGGGCCAAGATAAACGGA miR- C+AAG+TCACTAGTGGTT −0.2802 7.5575 0.56 5.63 SEQ ID NO: 315 224RP2# SEQ ID NO: 316 miR-296 miR-296GSP9# CATGATCAGCTGGGCCAAGAACAGGATTG miR- A+GG+GCCCCCCCTCAA −0.3178 8.3856 0.10 0.96 SEQ ID NO: 317 296RP2# SEQ ID NO: 318 miR-299 miR-299GSP9# CATGATCAGCTGGGCCAAGAATGTATGTG miR-299RP# T+GG+TTTACCGTCCC −0.3155 7.9383 1.30 12.96 SEQ ID NO: 319 SEQ ID NO: 320 miR-301 miR-301GSP CATGATCAGCTGGGCCAAGAGCTTTGACAA miR-301RP C+AG+TGCAATAGTATTGT −0.2839 8.314 2.55 25.52 SEQ ID NO: 321 SEQ ID NO: 322 miR- miR-302a*GSP CATGATCAGCTGGGCCAAGAAAAGCAAGTA miR- TAAA+CG+TGGATGTAC −0.2608 8.3921 0.04 0.41 302a* SEQ ID NO: 323 302a*RP SEQ ID NO: 324 miR-302a miR-302aGSP CATGATCAGCTGGGCCAAGATCACCAAAAC miR-302aRP T+AAG+TGCTTCCATGT −0.2577 9.6657 2.17 21.67 SEQ ID NO: 325 SEQ ID NO: 326 miR- miR-302b*GSP CATGATCAGCTGGGCCAAGAAGAAAGCACT miR- A+CTTTAA+CATGGAAGTG −0.2702 8.5153 0.02 0.24 302b* SEQ ID NO: 327 302b*RP SEQ ID NO: 328 miR-302b miR-302bGSP CATGATCAGCTGGGCCAAGACTACTAAAAC miR-302bRP T+AAG+TGCTTCCATGT −0.2398 9.1459 5.11 51.11 SEQ ID NO: 329 SEQ ID NO: 330 miR-302d miR-302dGSP CATGATCAGCTGGGCCAAGAACACTCAAAC miR-302dRP T+AAG+TGCTTCCATGT −0.2368 8.5602 5.98 59.78 SEQ ID NO: 331 SEQ ID NO: 332 miR- miR- CATGATCAGCTGGGCCAAGACAGCAGGTA miR- TT+TAA+CAT+GGGGGTACC −0.312 8.2904 0.33 3.28 302c* 302c*_GSP9# SEQ ID NO: 333 302c*_RP2# SEQ ID NO: 334 miR-302c miR- CATGATCAGCTGGGCCAAGACCACTGAAA miR- T+AAG+TGCTTCCATGTTTCA −0.2945 8.381 14.28 142.76 302cGSP9# SEQ ID NO: 335 302cRP5# SEQ ID NO: 336 miR-320 miR- CATGATCAGCTGGGCCAAGATTCGCCCT miR- AAAA+GCT+GGGTTGAGAGG −0.2677 7.8956 6.73 67.29 320_GSP8# SEQ ID NO: 337 320_RP3# SEQ ID NO: 338 miR-323 miR-323GSP CATGATCAGCTGGGCCAAGAAGAGGTCGAC miR-323RP G+CA+CATTACACGGT −0.2878 8.2546 0.19 1.92 SEQ ID NO: 339 SEQ ID NO: 340 miR-324- miR-324- CATGATCAGCTGGGCCAAGACCAGCAGCAC miR-324- C+CA+CTGCCCCAGGT −0.2698 8.5223 2.54 25.41 3p 3pGSP SEQ ID NO: 341 3pRP SEQ ID NO: 342 miR-324- miR-324- CATGATCAGCTGGGCCAAGAACACCAATGC miR-324- C+GC+ATCCCCTAGGG −0.2861 7.6865 0.06 0.62 5p 5pGSP SEQ ID NO: 343 5pRP SEQ ID NO: 344 miR-325 miR-325GSP CATGATCAGCTGGGCCAAGAACACTTACTG miR-325RP C+CT+AGTAGGTGTCC −0.2976 8.1925 0.01 0.14 SEQ ID NO: 345 SEQ ID NO: 346 miR-326 miR-326GSP CATGATCAGCTGGGCCAAGACTGGAGGAAG miR-326RP C+CT+CTGGGCCCTTC −0.2806 7.897 0.59 5.87 SEQ ID NO: 347 SEQ ID NO: 348 miR-328 miR-328GSP CATGATCAGCTGGGCCAAGAACGGAAGGGC miR-328RP C+TG+GCCCTCTCTGC −0.293 7.929 3.17 31.69 SEQ ID NO: 349 SEQ ID NO: 350 miR-330 miR-330GSP CATGATCAGCTGGGCCAAGATCTCTGCAGG miR-330RP G+CA+AAGCACACGGC −0.3009 7.7999 0.13 1.30 SEQ ID NO: 351 SEQ ID NO: 352 miR-331 miR-331GSP CATGATCAGCTGGGCCAAGATTCTAGGATA miR-331RP G+CC+CCTGGGCCTAT −0.2816 8.1643 0.45 4.54 SEQ ID NO: 353 SEQ ID NO: 354 miR-337 miR-337GSP CATGATCAGCTGGGCCAAGAAAAGGCATCA miR-337RP T+CC+AGCTCCTATATG −0.2968 8.7313 0.10 1.02 SEQ ID NO: 355 SEQ ID NO: 356 miR-338 miR-338GSP CATGATCAGCTGGGCCAAGATCAACAAAAT miR-338RP2 T+CC+AGCATCAGTGATTT −0.2768 8.5618 0.52 5.17 SEQ ID NO: 357 SEQ ID NO: 358 miR-339 miR-339GSP9# CATGATCAGCTGGGCCAAGATGAGCTCCT miR- T+CC+CTGTCCTCCAGG −0.303 8.4873 0.27 2.72 SEQ ID NO: 359 339RP2# SEQ ID NO: 360 miR-340 miR-340GSP CATGATCAGCTGGGCCAAGAGGCTATAAAG miR-340RP TC+CG+TCTCAGTTAC −0.2846 9.6673 0.15 1.45 SEQ ID NO: 361 SEQ ID NO: 362 miR-342 miR-342GSP3 CATGATCAGCTGGGCCAAGAGACGGGTG miR-342RP T+CT+CACACAGAAATCG −0.293 8.1553 4.69 46.85 SEQ ID NO: 363 SEQ ID NO: 364 miR-345 miR-345GSP CATGATCAGCTGGGCCAAGAGCCCTGGACT miR-345RP T+GC+TGACTCCTAGT −0.2909 8.468 0.04 0.40 SEQ ID NO: 365 SEQ ID NO: 366 miR-346 miR-346GSP CATGATCAGCTGGGCCAAGAAGAGGCAGGC miR-346RP T+GT+CTGCCCGCATG −0.2959 8.1958 0.25 2.54 SEQ ID NO: 367 SEQ ID NO: 368 miR-363 miR-363 CATGATCAGCTGGGCCAAGATACAGATGGA miR-363RP# AAT+TG+CAC+GGTATCC −0.2362 8.9762 0.44 4.36 GSP10# SEQ ID NO: 369 SEQ ID NO: 370 miR-367 miR-367GSP CATGATCAGCTGGGCCAAGATCACCATTGC miR-367RP AAT+TG+CACTTTAGCAAT −0.2819 8.6711 0.00 0.03 SEQ ID NO: 371 SEQ ID NO: 372 miR-368 miR-368GSP CATGATCAGCTGGGCCAAGAAAACGTGGAA miR-368RP2 A+CATAGA+GGAAATTCCAC −0.2953 8.0067 6.01 60.11 SEQ ID NO: 373 SEQ ID NO: 374 miR-370 miR-370GSP CATGATCAGCTGGGCCAAGACCAGGTTCCA miR-370RP G+CC+TGCTGGGGTGG −0.2825 8.3162 1.45 14.55 SEQ ID NO: 375 SEQ ID NO: 376 miR-371 miR-371GSP CATGATCAGCTGGGCCAAGAACACTCAAAA miR-371RP G+TG+CCGCCATCTTT −0.295 7.8812 2.51 25.12 SEQ ID NO: 377 SEQ ID NO: 378 miR-372 miR-372GSP CATGATCAGCTGGGCCAAGAACGCTCAAAT miR-372RP A+AA+GTGCTGCGACA −0.2984 8.9183 0.05 0.53 SEQ ID NO: 379 SEQ ID NO: 380 miR-373* miR-373*GSP CATGATCAGCTGGGCCAAGAGGAAAGCGCC miR-373*RP A+CT+CAAAATGGGGG −0.2705 8.4513 0.20 1.99 SEQ ID NO: 381 SEQ ID NO: 382 miR-373 miR-373GSP CATGATCAGCTGGGCCAAGAACACCCCAAA miR-373RP2 GA+AG+TGCTTCGATTTTGG −0.307 7.9056 9.13 91.32 SEQ ID NO: 383 SEQ ID NO: 384 miR-374 miR-374GSP2 CATGATCAGCTGGGCCAAGACACTTATCA miR-374RP TT+AT+AATA+CAACCTGATA −0.2655 9.3795 9.16 91.60 SEQ ID NO: 385 AG SEQ ID NO: 386 miR-375 miR-375GSP CATGATCAGCTGGGCCAAGATCACGCGAGC miR-375RP TT+TG+TTCGTTCGGC −0.3041 8.1181 0.09 0.90 SEQ ID NO: 387 SEQ ID NO: 388 miR-376b miR-376b CATGATCAGCTGGGCCAAGAAACATGGA miR- AT+CAT+AGA+GGAAAATCCA −0.2934 9.0188 1.07 10.74 GSP8# SEQ ID NO: 389 376bRP# SEQ ID NO: 390 miR-378 miR-378GSP CATGATCAGCTGGGCCAAGAACACAGGACC miR-378RP C+TC+CTGACTCCAGG −0.2899 8.1467 0.07 0.73 SEQ ID NO: 391 SEQ ID NO: 392 miR-379 miR- CATGATCAGCTGGGCCAAGATACGTTC miR- T+GGT+AGACTATGGAACG −0.2902 8.2149 10.89 108.86 379_GSP7# SEQ ID NO: 393 379RP2# SEQ ID NO: 394 miR-380- miR-380- CATGATCAGCTGGGCCAAGAGCGCATGTTC miR-380- T+GGT+TGACCATAGA −0.2462 9.4324 1.30 13.04 5p 5pGSP SEQ ID NO: 395 5pRP SEQ ID NO: 396 miR-380- miR-380- CATGATCAGCTGGGCCAAGAAAGATGTGGA miR-380- TA+TG+TAATATGGTCCACA −0.3037 8.0356 3.69 36.89 3p 3pGSP SEQ ID NO: 397 3pRP SEQ ID NO: 398 miR-381 miR-381GSP2 CATGATCAGCTGGGCCAAGAACAGAGAGC miR-381RP2 TATA+CAA+GGGCAAGCT −0.3064 8.8704 1.72 17.16 SEQ ID NO: 399 SEQ ID NO: 400 miR-382 miR-382GSP CATGATCAGCTGGGCCAAGACGAATCCACC miR-382RP G+AA+GTTGTTCGTGGT −0.2803 7.6738 0.66 6.57 SEQ ID NO: 401 SEQ ID NO: 402 miR-383 miR-383GSP CATGATCAGCTGGGCCAAGAAGCCACAATC miR-383RP2 A+GATC+AGAAGGTGATTGT −0.2866 8.1463 0.54 5.45 SEQ ID NO: 403 SEQ ID NO: 404 miR-410 miR-410 CATGATCAGCTGGGCCAAGAACAGGCCAT miR-410RP# AA+TA+TAA+CA+CAGATGGC −0.2297 8.5166 4.27 42.71 GSP9# SEQ ID NO: 405 SEQ ID NO: 406 miR-412 miR-412 CATGATCAGCTGGGCCAAGAACGGCTAGTG miR-412RP# A+CTT+CACCTGGTCCACTA −0.3001 7.9099 4.24 42.37 GSP10# SEQ ID NO: 407 SEQ ID NO: 408 miR-422a miR-422aGSP CATGATCAGCTGGGCCAAGAGGCCTTCTGA miR-422aRP C+TG+GACTTAGGGTC −0.3079 9.3108 5.95 59.54 SEQ ID NO: 409 SEQ ID NO: 410 miR-422b miR-422bGSP CATGATCAGCTGGGCCAAGAGGCCTTCTGA miR-422bRP C+TG+GACTTGGAGTC −0.2993 8.9437 4.86 48.56 SEQ ID NO: 411 SEQ ID NO: 412 miR-423 miR-423GSP CATGATCAGCTGGGCCAAGACTGAGGGGCC miR-423RP A+GC+TCGGTCTGAGG −0.3408 9.2274 6.06 60.62 SEQ ID NO: 413 SEQ ID NO: 414 miR-424 miR-424GSP# CATGATCAGCTGGGCCAAGATTCAAAACAT miR- C+AG+CAGCAATTCATGTTTT −0.3569 9.3419 10.78 107.85 SEQ ID NO: 415 424RP2# SEQ ID NO: 416 miR-425 miR-425GSP CATGATCAGCTGGGCCAAGAGGCGGACACG miR-425RP A+TC+GGGAATGTCGT −0.2932 7.9786 0.39 3.93 SEQ ID NO: 417 SEQ ID NO: 418 miR-429 miR- CATGATCAGCTGGGCCAAGAACGGTTTTACC miR- T+AATAC+TG+TCTGGTAAAA −0.2458 8.2805 16.21 162.12 429_GSP11# SEQ ID NO: 419 429RP5# SEQ ID NO: 420 miR-431 miR-431 CATGATCAGCTGGGCCAAGATGCATGACGG miR-431RP# T+GT+CTTGCAGGCCG −0.3107 7.7127 7.00 70.05 GSP10# SEQ ID NO: 421 SEQ ID NO: 422 miR-448 miR-448GSP CATGATCAGCTGGGCCAAGAATGGGACATC miR-448RP TTG+CATA+TGTAGGATG −0.3001 8.4969 0.12 1.16 SEQ ID NO: 423 SEQ ID NO: 424 miR-449 miR- CATGATCAGCTGGGCCAAGAACCAGCTAAC miR- T+GG+CAGTGTATTGTTAGC −0.3225 8.4953 2.57 25.70 449GSP10# SEQ ID NO: 425 449RP2# SEQ ID NO: 426 miR-450 miR-450GSP CATGATCAGCTGGGCCAAGATATTAGGAAC miR-450RP TTTT+TG+CGATGTGTT −0.2906 8.1404 0.48 4.82 SEQ ID NO: 427 SEQ ID NO: 428 miR-451 miR-451 CATGATCAGCTGGGCCAAGAAAACTCAGTA miR-451RP# AAA+CCG+TTA+CCATTACTGA −0.2544 8.0291 1.73 17.35 GSP10# SEQ ID NO: 429 SEQ ID NO: 430 let7a let7a-GSP2# CATGATCAGCTGGGCCAAGAAACTATAC let7a-RP# T+GA+GGTAGTAGGTTG −0.3089 9.458 0.04 0.38 SEQ ID NO: 431 SEQ ID NO: 432 let7b let7b-GSP2# CATGATCAGCTGGGCCAAGAAACCACAC let7b-RP# T+GA+GGTAGTAGGTTG −0.2978 7.9144 0.05 0.54 SEQ ID NO: 433 SEQ ID NO: 432 let7c let7c-GSP2# CATGATCAGCTGGGCCAAGAAACCATAC let7c-RP# T+GA+GGTAGTAGGTTG −0.308 7.9854 0.01 0.14 SEQ ID NO: 434 SEQ ID NO: 432 let7d let7d-GSP2# CATGATCAGCTGGGCCAAGAACTATGCA let7d-RP# A+GA+GGTAGTAGGTTG −0.3238 8.3359 0.06 0.57 SEQ ID NO: 435 SEQ ID NO: 436 let7e let7e-GSP2# CATGATCAGCTGGGCCAAGAACTATACA let7e-RP# T+GA+GGTAGGAGGTTG −0.3284 9.7594 0.22 2.20 SEQ ID NO: 437 SEQ ID NO: 438 let7f let7f-GSP2# CATGATCAGCTGGGCCAAGAAACTATAC let7f-RP# T+GA+GGTAGTAGATTG −0.2901 11.107 0.32 3.18 SEQ ID NO: 439 SEQ ID NO: 440 let7g let7g-GSP2# CATGATCAGCTGGGCCAAGAACTGTACA let7g-RP# T+GA+GGTAGTAGTTTG −0.3469 9.8235 0.16 1.64 SEQ ID NO: 441 SEQ ID NO: 442 let7i let7i-GSP2# CATGATCAGCTGGGCCAAGAACAGCACA let7i-RP# T+GA+GGTAGTAGTTTG −0.321 10.82 0.20 1.99 SEQ ID NO: 443 SEQ ID NO: 444 miR-377 miR-377GSP CATGATCAGCTGGGCCAAGAACAAAAGTTG miR-377RP2 AT+CA+CACAAAGGCAAC −0.2979 10.612 13.45 134.48 SEQ ID NO: 445 SEQ ID NO: 446 miR-376a miR- CATGATCAGCTGGGCCAAGAACGTGGA miR- AT+CAT+AGA+GGAAAATCC −0.2938 10.045 63.00 630.00 376a_GSP7 SEQ ID NO: 447 376a_RP5 SEQ ID NO: 448 miR-22 miR-22GSP CATGATCAGCTGGGCCAAGAACAGTTCTTC miR-22RP A+AG+CTGCCAGTTGA −0.2862 8.883 20.46 204.58 SEQ ID NO: 449 SEQ ID NO: 450 miR-200c miR-200cGSP2 CATGATCAGCTGGGCCAAGACCATCATTA miR-200cRP T+AA+TACTGCCGGGT −0.3094 11.5 15.99 159.91 SEQ ID NO: 451 SEQ ID NO: 452 miR-24 miR-24GSP CATGATCAGCTGGGCCAAGACTGTTCCTGC miR-24RP T+GG+CTCAGTTCAGC −0.3123 8.6824 24.34 243.38 SEQ ID NO: 453 SEQ ID NO: 454 miR- miR-29cGSP10 CATGATCAGCTGGGCCAAGAACCGATTTCA miR-29cRP T+AG+CACCATTTGAAAT −0.2975 8.8441 23.22 232.17 29cDNA SEQ ID NO: 455 SEQ ID NO: 456 miR-18 miR-18GSP CATGATCAGCTGGGCCAAGATATCTGCACT miR-18RP T+AA+GGTGCATCTAGT −0.3209 9.0999 14.90 149.01 SEQ ID NO: 457 SEQ ID NO: 458 miR-185 miR-185GSP CATGATCAGCTGGGCCAAGAGAACTGCCTT miR-185RP T+GG+AGAGAAAGGCA −0.3081 8.9289 15.73 157.32 SEQ ID NO: 459 SEQ ID NO: 460 miR-181b miR- CATGATCAGCTGGGCCAAGACCCACCGA miR- AA+CATT+CATTGCTGTC −0.3115 10.846 15.87 158.67 181bGSP8# SEQ ID NO: 461 181bRP2# SEQ ID NO: 462 miR-128a miR-128aGSP CATGATCAGCTGGGCCAAGAAAAAGAGACC miR- TCACAGTGAACCGGT approx. approx. approx. approx. SEQ ID NO: 161 128anLRP SEQ ID NO: 494 −0.2866 8.0867 0.16 1.60 miR-138 miR-138GSP2 CATGATCAGCTGGGCCAAGACGGCCTGAT miR- AGCTGGTGTTGTGAA approx. approx. approx. approx. SEQ ID NO: 187 138nLRP SEQ ID NO: 495 −0.3023 9.0814 0.22 2.19 miR-143 miR-143GSP8# CATGATCAGCTGGGCCAAGATGAGCTAC miR- TGAGATGAAGCACTGT approx. approx. approx. approx. SEQ ID NO: 197 143nLRP SEQ ID NO: 496 −0.3008 9.2675 0.37 3.71 miR-150 miR-150GSP3 CATGATCAGCTGGGCCAAGACACTGGTA miR- TCTCCCAACCCTTGTA approx. approx. approx. approx. SEQ ID NO: 213 150nLRP SEQ ID NO: 497 −0.2943 8.3945 0.06 0.56 miR-181a miR- CATGATCAGCTGGGCCAAGAACTCACCGA miR- AACATTCAACGCTGT approx. approx. approx. approx. 181aGSP9# SEQ ID NO: 227 181anLRP SEQ ID NO: 498 −0.2919 7.968 1.70 17.05 miR-194 mir194GSP8# CATGATCAGCTGGGCCAAGATCCACATG miR- TGTAACAGCAACTCCA approx. approx. approx. approx. SEQ ID NO: 255 194nLRP SEQ ID NO: 499 −0.3078 8.8045 0.37 3.69 #denotes primers for assays that required extensive testing and primer design modification to achieve optimal assay results including high sensitivity and high dynamic range.

Example 4

This Example describes assays and primers designed for quantitative analysis of murine miRNA expression patterns.

Methods: The representative murine microRNA target templates described in TABLE 7 are publicly available accessible on the World Wide Web at the Wellcome Trust Sanger Institute website in the “miRBase sequence database” as described in Griffith-Jones et al. (2004), Nucleic Acids Research 32:D109-D111 and Griffith-Jones et al. (2006), Nucleic Acids Research 34: D140-D144. As indicated below in TABLE 7, the murine microRNA templates are either totally identical to the corresponding human microRNA templates, identical in the overlapping sequence with differing ends, or contain one or more base pair changes as compared to the human microRNA sequence. The murine microRNA templates that are identical or that have identical overlapping sequence to the corresponding human templates can be assayed using the same primer sets designed for the human microRNA templates, as indicated in TABLE 7. For the murine microRNA templates with one or more base pair changes in comparison to the corresponding human templates, primer sets have been designed specifically for detection of the murine microRNA, and these primers are provided in TABLE 7. The extension primer reaction and quantitative PCR reactions for detection of the murine microRNA templates may be carried out as described in EXAMPLE 3.

TABLE 7 PRIMERS TO DETECT MURINE MICRORNA TARGET TEMPLATES Mouse Target Mouse microRNA as compared microRNA: Extension Primer Name Extension Primer Sequence Reverse Primer Name Reverse Primer Sequence to Human microRNA miR-1 miR1GSP10 CATGATCAGCTGGGCCAAGATACATACTTC miR-1RP T+G+GAA+TG+TAAAGAAGT Identical SEQ ID NO: 47 SEQ ID NO: 48 miR-7 miR-7GSP10 CATGATCAGCTGGGCCAAGAAACAAAATC miR-7_RP6 T+GGAA+GACTTGTGATTTT one or more base pairs differ SEQ ID NO: 486 SEQ ID NO: 487 miR-9* miR-9*GSP CATGATCAGCTGGGCCAAGAACTTTCGGTT miR-9*RP TAAA+GCT+AGATAACCG Identical overlapping sequence, SEQ ID NO: 51 SEQ ID NO: 52 ends differ miR-10a miR-10aGSP CATGATCAGCTGGGCCAAGACACAAATTCG miR-10aRP T+AC+CCTGTAGATCCG Identical SEQ ID NO: 53 SEQ ID NO: 54 miR-10b miR-10b_GSP11 CATGATCAGCTGGGCCAAGAACACAAATTCG miR-10b_RP2 C+CC+TGT+AGAACCGAAT one or more base pairs differ SEQ ID NO: 492 SEQ ID NO: 493 miR-15a miR-15aGSP CATGATCAGCTGGGCCAAGACACAAACCAT miR-15aRP T+AG+CAGCACATAATG Identical SEQ ID NO: 57 SEQ ID NO: 58 miR-15b miR-15bGSP2 CATGATCAGCTGGGCCAAGATGTAAACCA miR-15bRP T+AG+CAGCACATCAT Identical SEQ ID NO: 59 SEQ ID NO: 60 miR-16 miR-16GSP2 CATGATCAGCTGGGCCAAGACGCCAATAT miR-16RP T+AG+CAGCACGTAAA Identical SEQ ID NO: 61 SEQ ID NO: 62 miR-17-3p miR-17-3pGSP CATGATCAGCTGGGCCAAGAACAAGTGCCC miR-17-3pRP A+CT+GCAGTGAGGGC one or more base pairs differ SEQ ID NO: 463 SEQ ID NO: 464 miR-17-5p miR-17-5pGSP2 CATGATCAGCTGGGCCAAGAACTACCTGC miR-17-5pRP C+AA+AGTGCTTACAGTG Identical SEQ ID NO: 65 SEQ ID NO: 66 miR-19a miR-19aGSP2 CATGATCAGCTGGGCCAAGATCAGTTTTG miR-19aRP TG+TG+CAAATCTATGC Identical SEQ ID NO: 67 SEQ ID NO: 68 miR-19b miR-19bGSP CATGATCAGCTGGGCCAAGATCAGTTTTGC miR-19bRP TG+TG+CAAATCCATG Identical SEQ ID NO: 69 SEQ ID NO: 70 miR-20 miR-20GSP3 CATGATCAGCTGGGCCAAGACTACCTGC miR-20RP T+AA+AGTGCTTATAGTGCA Identical SEQ ID NO: 71 SEQ ID NO: 72 miR-21 miR-21GSP2 CATGATCAGCTGGGCCAAGATCAACATCA miR-21RP T+AG+CTTATCAGACTGATG Identical SEQ ID NO: 73 SEQ ID NO: 74 miR-23a miR-23aGSP CATGATCAGCTGGGCCAAGAGGAAATCCCT miR-23aRP A+TC+ACATTGCCAGG Identical SEQ ID NO: 75 SEQ ID NO: 76 miR-23b miR-23bGSP CATGATCAGCTGGGCCAAGAGGTAATCCCT miR-23bRP A+TC+ACATTGCCAGG Identical SEQ ID NO: 77 SEQ ID NO: 78 miR-24 miR-24P5 CATGATCAGCTGGGCCAAGACTGTTCCTGC miR24-1,2R TGG+CTCAGTTCAGC Identical TG SEQ ID NO: 19 SEQ ID NO: 7 miR-25 miR-25GSP CATGATCAGCTGGGCCAAGATCAGACCGAG miR-25RP C+AT+TGCACTTGTCTC Identical SEQ ID NO: 79 SEQ ID NO: 80 miR-26a miR-26aGSP9 CATGATCAGCTGGGCCAAGAGCCTATCCT miR-26aRP2 TT+CA+AGTAATCCAGGAT Identical SEQ ID NO: 81 SEQ ID NO: 82 miR-26b miR-26bGSP9 CATGATCAGCTGGGCCAAGAAACCTATCC miR-26bRP2 TT+CA+AGT+AATTCAGGAT Identical SEQ ID NO: 83 SEQ ID NO: 84 miR-27a miR-27aGSP CATGATCAGCTGGGCCAAGAGCGGAACTTA miR-27aRP TT+CA+CAGTGGCTAA Identical SEQ ID NO: 85 SEQ ID NO: 86 miR-27b miR-27bGSP CATGATCAGCTGGGCCAAGAGCAGAACTTA miR-27bRP TT+CA+CAGTGGCTAA Identical SEQ ID NO: 87 SEQ ID NO: 88 miR-28 miR-28GSP CATGATCAGCTGGGCCAAGACTCAATAGAC miR-28RP A+AG+GAGCTCACAGT Identical SEQ ID NO: 89 SEQ ID NO: 90 miR-29a miR-29aGSP8 CATGATCAGCTGGGCCAAGAAACCGATT miR-29aRP2 T+AG+CACCATCTGAAAT Identical SEQ ID NO: 91 SEQ ID NO: 92 miR-29b miR-29bGSP2 CATGATCAGCTGGGCCAAGAAACACTGAT miR-29bRP2 T+AG+CACCATTTGAAATCAG Identical SEQ ID NO: 93 SEQ ID NO: 94 miR-30a-5p miR-30a-5pGSP CATGATCAGCTGGGCCAAGACTTCCAGTCG miR-30a-5pRP T+GT+AAACATCCTCGAC Identical SEQ ID NO: 95 SEQ ID NO: 96 miR-30b miR-30bGSP CATGATCAGCTGGGCCAAGAAGCTGAGTGT miR-30bRP TGT+AAA+CATCCTACACT Identical SEQ ID NO: 97 SEQ ID NO: 98 miR-30c miR-30cGSP CATGATCAGCTGGGCCAAGAGCTGAGAGTG miR-30cRP TGT+AAA+CATCCTACACT Identical SEQ ID NO: 99 SEQ ID NO: 100 miR-30d miR-30dGSP CATGATCAGCTGGGCCAAGACTTCCAGTCG miR-30dRP T+GTAAA+CATCCCCG Identical SEQ ID NO: 101 SEQ ID NO: 102 miR-30e-3p miR-30e-3pGSP9 CATGATCAGCTGGGCCAAGAGCTGTAAAC miR-30e-3pRP5 CTTT+CAGT+CGGATGTTT Identical SEQ ID NO: 103 SEQ ID NO: 104 miR-31 miR-31GSP CATGATCAGCTGGGCCAAGACAGCTATGCC miR-31RP G+GC+AAGATGCTGGC Identical overlapping sequence, SEQ ID NO: 107 SEQ ID NO: 108 ends differ miR-32 miR-32GSP CATGATCAGCTGGGCCAAGAGCAACTTAGT miR-32RP TATTG+CA+CATTACTAAG Identical SEQ ID NO: 109 SEQ ID NO: 110 miR-33 miR-33GSP2 CATGATCAGCTGGGCCAAGACAATGCAAC miR-33RP G+TG+CATTGTAGTTGC Identical SEQ ID NO: 111 SEQ ID NO: 112 miR-34a miR-34aGSP CATGATCAGCTGGGCCAAGAAACAACCAGC miR-34aRP T+GG+CAGTGTCTTAG Identical SEQ ID NO: 113 SEQ ID NO: 114 miR-34b miR-34bGSP CATGATCAGCTGGGCCAAGACAATCAGCTA miR-34bRP TA+GG+CAGTGTAATT one or more base pairs differ SEQ ID NO: 115 SEQ ID NO: 482 miR-34c miR-34cGSP CATGATCAGCTGGGCCAAGAGCAATCAGCT miR-34cRP A+GG+CAGTGTAGTTA Identical SEQ ID NO: 117 SEQ ID NO: 118 miR-92 miR-92GSP CATGATCAGCTGGGCCAAGACAGGCCGGGA miR-92RP T+AT+TGCACTTGTCCC Identical SEQ ID NO: 119 SEQ ID NO: 120 miR-93 miR-93GSP CATGATCAGCTGGGCCAAGACTACCTGCAC miR-93RP AA+AG+TGCTGTTCGT Identical overlapping sequence, SEQ ID NO: 121 SEQ ID NO: 122 ends differ miR-96 miR-96GSP CATGATCAGCTGGGCCAAGAGCAAAAATGT miR-96RP T+TT+GGCACTAGCAC Identical overlapping sequence, SEQ ID NO: 125 SEQ ID NO: 126 ends differ miR-98 miR-98GSP CATGATCAGCTGGGCCAAGAAACAATACAA miR-98RP TGA+GGT+AGTAAGTTG Identical SEQ ID NO: 127 SEQ ID NO: 128 miR-99a miR-99aGSP CATGATCAGCTGGGCCAAGACACAAGATCG miR-99aRP A+AC+CCGTAGATCCG Identical overlapping sequence, SEQ ID NO: 129 SEQ ID NO: 130 ends differ miR-99b miR-99bGSP CATGATCAGCTGGGCCAAGACGCAAGGTCG miR-99bRP C+AC+CCGTAGAACCG Identical SEQ ID NO: 131 SEQ ID NO: 132 miR-100 miR-100GSP CATGATCAGCTGGGCCAAGACACAAGTTCG miR-100RP A+AC+CCGTAGATCCG Identical SEQ ID NO: 133 SEQ ID NO: 134 miR-101 miR-101GSP CATGATCAGCTGGGCCAAGACTTCAGTTAT miR-101RP TA+CAG+TACTGTGATAACT Identical SEQ ID NO: 135 SEQ ID NO: 136 miR-103 miR-103GSP CATGATCAGCTGGGCCAAGATCATAGCCCT miR-103RP A+GC+AGCATTGTACA Identical SEQ ID NO: 137 SEQ ID NO: 138 miR-106a miR-106aGSP CATGATCAGCTGGGCCAAGATACCTGCAC miR-106aRP CAA+AG+TGCTAACAGTG one or more base pairs differ SEQ ID NO: 472 SEQ ID NO: 473 miR-106b miR-106bGSP CATGATCAGCTGGGCCAAGAATCTGCACTG miR-106bRP T+AAAG+TGCTGACAGT Identical SEQ ID NO: 143 SEQ ID NO: 144 miR-107 miR-107GSP8 CATGATCAGCTGGGCCAAGATGATAGCC miR-107RP2 A+GC+AGCATTGTACAG Identical SEQ ID NO: 145 SEQ ID NO: 146 miR-122a miR-122aGSP CATGATCAGCTGGGCCAAGAACAAACACCA miR-122aRP T+GG+AGTGTGACAAT Identical SEQ ID NO: 147 SEQ ID NO: 148 miR-124a miR-124aGSP CATGATCAGCTGGGCCAAGATGGCATTCAC miR-124aRP T+TA+AGGCACGCGGT Identical overlapping sequence, SEQ ID NO: 149 SEQ ID NO: 150 ends differ miR-125a miR-125aGSP CATGATCAGCTGGGCCAAGACACAGGTTAA miR-125aRP T+CC+CTGAGACCCTT Identical SEQ ID NO: 151 SEQ ID NO: 152 miR-125b miR-125bGSP CATGATCAGCTGGGCCAAGATCACAAGTTA miR-125bRP T+CC+CTGAGACCCTA Identical SEQ ID NO: 153 SEQ ID NO: 154 miR-126 miR-126GSP CATGATCAGCTGGGCCAAGAGCATTATTAC miR-126RP T+CG+TACCGTGAGTA Identical SEQ ID NO: 155 SEQ ID NO: 156 miR-126* miR-126*GSP3 CATGATCAGCTGGGCCAAGACGCGTACC miR-126*RP C+ATT+ATTA+CTTTTGGTACG Identical SEQ ID NO: 157 SEQ ID NO: 158 miR-127 miR-127GSP CATGATCAGCTGGGCCAAGAAGCCAAGCTC miR-127RP T+CG+GATCCGTCTGA Identical overlapping sequence, SEQ ID NO: 159 SEQ ID NO: 160 ends differ miR-128a miR-128aGSP CATGATCAGCTGGGCCAAGAAAAAGAGACC miR-128aRP T+CA+CAGTGAACCGG Identical SEQ ID NO: 161 SEQ ID NO: 162 miR-128b miR-128bGSP CATGATCAGCTGGGCCAAGAGAAAGAGACC miR-128bRP T+CA+CAGTGAACCGG Identical SEQ ID NO: 163 SEQ ID NO: 164 miR-130a miR-130aGSP CATGATCAGCTGGGCCAAGAATGCCCTTTT miR-130aRP C+AG+TGCAATGTTAAAAG Identical SEQ ID NO: 167 SEQ ID NO: 168 miR-130b miR-130bGSP CATGATCAGCTGGGCCAAGAATGCCCTTTC miR-130bRP C+AG+TGCAATGATGA Identical SEQ ID NO: 169 SEQ ID NO: 170 miR-132 miR-132GSP CATGATCAGCTGGGCCAAGACGACCATGGC miR-132RP T+AA+CAGTCTACAGCC Identical SEQ ID NO: 171 SEQ ID NO: 172 miR-133a miR-133aGSP CATGATCAGCTGGGCCAAGAACAGCTGGTT miR-133aRP T+TG+GTCCCCTTCAA Identical SEQ ID NO: 173 SEQ ID NO: 174 miR-133b miR-133bGSP CATGATCAGCTGGGCCAAGATAGCTGGTTG miR-133bRP T+TG+GTCCCCTTCAA Identical SEQ ID NO: 175 SEQ ID NO: 176 miR-134 miR-134GSP CATGATCAGCTGGGCCAAGACCCTCTGGTC miR-134RP T+GT+GACTGGTTGAC Identical overlapping sequence, SEQ ID NO: 177 SEQ ID NO: 178 ends differ miR-135a miR-135aGSP CATGATCAGCTGGGCCAAGATCACATAGGA miR-135aRP T+AT+GGCTTTTTATTCCT Identical SEQ ID NO: 179 SEQ ID NO: 180 miR-135b miR-135bGSP CATGATCAGCTGGGCCAAGACACATAGGAA miR-135bRP T+AT+GGCTTTTCATTCC Identical SEQ ID NO: 181 SEQ ID NO: 182 miR-136 miR-136GSP CATGATCAGCTGGGCCAAGATCCATCATCA miR-136RP A+CT+CCATTTGTTTTGATG Identical SEQ ID NO: 183 SEQ ID NO: 184 miR-137 miR-137GSP CATGATCAGCTGGGCCAAGACTACGCGTAT miR-137RP T+AT+TGCTTAAGAATACGC Identical overlapping sequence, SEQ ID NO: 185 SEQ ID NO: 186 ends differ miR-138 miR-138GSP2 CATGATCAGCTGGGCCAAGACGGCCTGAT miR-138RP A+GC+TGGTGTTGTGA Identical SEQ ID NO: 187 SEQ ID NO: 188 miR-139 miR-139GSP CATGATCAGCTGGGCCAAGAAGACACGTGC miR-139RP T+CT+ACAGTGCACGT Identical SEQ ID NO: 189 SEQ ID NO: 190 miR-140 miR-140GSP CATGATCAGCTGGGCCAAGACTACCATAGG miR-140RP A+GT+GGTTTTACCCT Identical overlapping sequence, SEQ ID NO: 191 SEQ ID NO: 192 ends differ miR-141 miR-141GSP9 CATGATCAGCTGGGCCAAGACCATCTTTA miR-141RP2 TAA+CAC+TGTCTGGTAA Identical SEQ ID NO: 193 SEQ ID NO: 194 miR-142-3p miR-142-3pGSP3 CATGATCAGCTGGGCCAAGATCCATAAA miR-142-3pRP TGT+AG+TGTTTCCTACT Identical overlapping sequence, SEQ ID NO: 195 SEQ ID NO: 196 ends differ miR-143 miR-143GSP8 CATGATCAGCTGGGCCAAGATGAGCTAC miR-143RP2 T+GA+GATGAAGCACTG Identical SEQ ID NO: 197 SEQ ID NO: 198 miR-144 miR-144GSP2 CATGATCAGCTGGGCCAAGACTAGTACAT miR-144RP TA+CA+GTAT+AGATGATG Identical SEQ ID NO: 199 SEQ ID NO: 200 miR-145 miR-145GSP2 CATGATCAGCTGGGCCAAGAAAGGGATTC miR-145RP G+TC+CAGTTTTCCCA Identical SEQ ID NO: 201 SEQ ID NO: 202 miR-146 miR-146GSP3 CATGATCAGCTGGGCCAAGAAACCCATG miR-146RP T+GA+GAACTGAATTCCA Identical SEQ ID NO: 203 SEQ ID NO: 204 miR-148a miR-148aGSP2 CATGATCAGCTGGGCCAAGAACAAAGTTC miR-148aRP2 T+CA+GTGCACTACAGAACT Identical SEQ ID NO: 207 SEQ ID NO: 208 miR-148b miR-148bGSP2 CATGATCAGCTGGGCCAAGAACAAAGTTC miR-148bRP T+CA+GTGCATCACAG Identical SEQ ID NO: 209 SEQ ID NO: 210 miR-149 miR-149GSP2 CATGATCAGCTGGGCCAAGAGGAGTGAAG miR-149RP T+CT+GGCTCCGTGTC Identical SEQ ID NO: 211 SEQ ID NO: 212 miR-150 miR-150GSP3 CATGATCAGCTGGGCCAAGACACTGGTA miR-150RP T+CT+CCCAACCCTTG Identical SEQ ID NO: 213 SEQ ID NO: 214 miR-151 miR-151GSP2 CATGATCAGCTGGGCCAAGACCTCAAGGA miR-151RP A+CT+AGACTGAGGCTC one or more base pairs differ SEQ ID NO: 215 SEQ ID NO: 477 miR-152 miR-152GSP2 CATGATCAGCTGGGCCAAGACCCAAGTTC miR-152RP T+CA+GTGCATGACAG Identical SEQ ID NO: 217 SEQ ID NO: 218 miR-153 miR-153GSP2 CATGATCAGCTGGGCCAAGATCACTTTTG miR-153RP TTG+CAT+AGTCACAAAA Identical overlapping sequence, SEQ ID NO: 219 SEQ ID NO: 220 ends differ miR-154 miR-154GSP9 CATGATCAGCTGGGCCAAGACGAAGGCAA miR-154RP3 TA+GGTTA+TCCGTGTT Identical SEQ ID NO: 223 SEQ ID NO: 224 miR-155 miR-155GSP8 CATGATCAGCTGGGCCAAGACCCCTATC miR-155RP2 TT+AA+TGCTAATTGTGATAGG one or more base pairs differ SEQ ID NO: 225 SEQ ID NO: 489 miR-181a miR-181aGSP9 CATGATCAGCTGGGCCAAGAACTCACCGA miR-181aRP2 AA+CATT+CAACGCTGTC Identical SEQ ID NO: 227 SEQ ID NO: 228 miR-181c miR-181cGSP9 CATGATCAGCTGGGCCAAGAACTCACCGA miR-181cRP2 AA+CATT+CAACCTGTCG Identical SEQ ID NO: 229 SEQ ID NO: 230 miR-182 miR-182*GSP CATGATCAGCTGGGCCAAGATAGTTGGCAA miR-182*RP T+GG+TTCTAGACTTGC Identical SEQ ID NO: 231 SEQ ID NO: 232 miR-183 miR-183GSP2 CATGATCAGCTGGGCCAAGACAGTGAATT miR-183RP T+AT+GGCACTGGTAG Identical SEQ ID NO: 235 SEQ ID NO: 236 miR-184 miR-184GSP2 CATGATCAGCTGGGCCAAGAACCCTTATC miR-184RP T+GG+ACGGAGAACTG Identical SEQ ID NO: 237 SEQ ID NO: 238 miR-186 miR-186GSP9 CATGATCAGCTGGGCCAAGAAAGCCCAAA miR-186RP3 CA+AA+GAATT+CTCCTTTTGG Identical SEQ ID NO: 239 SEQ ID NO: 240 miR-187 miR-187GSP CATGATCAGCTGGGCCAAGACGGCTGCAAC miR-187RP T+CG+TGTCTTGTGTT Identical overlapping sequence, SEQ ID NO: 241 SEQ ID NO: 242 ends differ miR-188 miR-188GSP CATGATCAGCTGGGCCAAGAACCCTCCACC miR-188RP C+AT+CCCTTGCATGG Identical SEQ ID NO: 243 SEQ ID NO: 244 miR-189 miR-189GSP2 CATGATCAGCTGGGCCAAGAACTGATATC miR-189RP G+TG+CCTACTGAGCT Identical SEQ ID NO: 245 SEQ ID NO: 246 miR-190 miR-190GSP9 CATGATCAGCTGGGCCAAGAACCTAATAT miR-190RP4 T+GA+TA+TGTTTGATATATTAG Identical SEQ ID NO: 247 SEQ ID NO: 248 miR-191 miR-191GSP2 CATGATCAGCTGGGCCAAGAAGCTGCTTT miR-191RP2 C+AA+CGGAATCCCAAAAG Identical SEQ ID NO: 249 SEQ ID NO: 250 miR-192 miR-192GSP2 CATGATCAGCTGGGCCAAGAGGCTGTCAA miR-192RP C+TGA+CCTATGAATTGAC Identical overlapping sequence, SEQ ID NO: 251 SEQ ID NO: 252 ends differ miR-193 miR-193GSP9 CATGATCAGCTGGGCCAAGACTGGGACTT miR-193RP2 AA+CT+GGCCTACAAAG Identical SEQ ID NO: 253 SEQ ID NO: 254 miR-194 mir194GSP8 CATGATCAGCTGGGCCAAGATCCACATG mir194RP TG+TAA+CAGCAACTCCA Identical SEQ ID NO: 255 SEQ ID NO: 256 miR-195 miR-195GSP9 CATGATCAGCTGGGCCAAGAGCCAATATT miR-195RP3 T+AG+CAG+CACAGAAATA Identical SEQ ID NO: 257 SEQ ID NO: 258 miR-196a miR-196aGSP CATGATCAGCTGGGCCAAGACCAACAACAT miR-196aRP TA+GG+TAGTTTCATGTTG Identical SEQ ID NO: 261 SEQ ID NO: 262 miR-196b miR-196bGSP CATGATCAGCTGGGCCAAGACCAACAACAG miR-196bRP TA+GGT+AGTTTCCTGT Identical SEQ ID NO: 259 SEQ ID NO: 260 miR-199a* miR-199a*GSP2 CATGATCAGCTGGGCCAAGAAACCAATGT miR-199a*RP T+AC+AGTAGTCTGCAC Identical SEQ ID NO: 267 SEQ ID NO: 268 miR-199a miR-199aGSP2 CATGATCAGCTGGGCCAAGAGAACAGGTA miR-199aRP C+CC+AGTGTTCAGAC Identical SEQ ID NO: 269 SEQ ID NO: 270 miR-199b miR-199bGSP CATGATCAGCTGGGCCAAGAGAACAGGTAG miR-199bRP C+CC+AGTGTTTAGAC one or more base pairs differ SEQ ID NO: 475 SEQ ID NO: 272 miR-200a miR-200aGSP2 CATGATCAGCTGGGCCAAGAACATCGTTA miR-200aRP TAA+CAC+TGTCTGGT Identical SEQ ID NO: 273 SEQ ID NO: 274 miR-200b miR-200bGSP2 CATGATCAGCTGGGCCAAGAGTCATCATT miR-200bRP TAATA+CTG+CCTGGTAAT Identical SEQ ID NO: 275 SEQ ID NO: 276 miR-203 miR-203GSP2 CATGATCAGCTGGGCCAAGACTAGTGGTC miR-203RP G+TG+AAATGTTTAGGACC Identical overlapping sequence, SEQ ID NO: 279 SEQ ID NO: 280 ends differ miR-204 miR-204GSP2 CATGATCAGCTGGGCCAAGAAGGCATAGG miR-204RP T+TC+CCTTTGTCATCC Identical overlapping sequence, SEQ ID NO: 281 SEQ ID NO: 282 ends differ miR-205 miR-205GSP CATGATCAGCTGGGCCAAGACAGACTCCGG miR-205RP T+CCTT+CATTCCACC Identical SEQ ID NO: 283 SEQ ID NO: 284 miR-206 mir206GSP7 CATGATCAGCTGGGCCAAGACCACACA miR-206RP T+G+GAA+TGTAAGGAAGTGT Identical SEQ ID NO: 285 SEQ ID NO: 286 miR-208 miR-208_GSP13 CATGATCAGCTGGGCCAAGAACAAGCTTTT miR-208_RP4 ATAA+GA+CG+AGCAAAAAG Identical TGC SEQ ID NO: 288 SEQ ID NO: 287 miR-210 miR-210GSP CATGATCAGCTGGGCCAAGATCAGCCGCTG miR-210RP C+TG+TGCGTGTGACA Identical SEQ ID NO: 289 SEQ ID NO: 290 miR-211 miR-211GSP2 CATGATCAGCTGGGCCAAGAAGGCAAAGG miR-211RP T+TC+CCTTTGTCATCC one or more base pairs differ SEQ ID NO: 491 SEQ ID NO: 292 miR-212 miR-212GSP9 CATGATCAGCTGGGCCAAGAGGCCGTGAC miR-212RP2 T+AA+CAGTCTCCAGTCA Identical SEQ ID NO: 293 SEQ ID NO: 294 miR-213 miR-213GSP CATGATCAGCTGGGCCAAGAGGTACAATCA miR-213RP A+CC+ATCGACCGTTG Identical SEQ ID NO: 295 SEQ ID NO: 296 miR-214 miR-214GSP CATGATCAGCTGGGCCAAGACTGCCTGTCT miR-214RP A+CA+GCAGGCACAGA Identical SEQ ID NO: 297 SEQ ID NO: 298 miR-215 miR-215GSP2 CATGATCAGCTGGGCCAAGAGTCTGTCAA miR-215RP A+TGA+CCTATGATTTGAC one or more base pairs differ SEQ ID NO: 299 SEQ ID NO: 469 miR-216 miR-216GSP9 CATGATCAGCTGGGCCAAGACACAGTTGC mir216RP TAA+TCT+CAGCTGGCA Identical SEQ ID NO: 301 SEQ ID NO: 302 miR-217 miR-217GSP2 CATGATCAGCTGGGCCAAGAATCCAGTCA miR-217RP2 T+AC+TGCATCAGGAACTGA one or more base pairs differ SEQ ID NO: 481 SEQ ID NO: 304 miR-218 miR-218GSP2 CATGATCAGCTGGGCCAAGAACATGGTTA miR-218RP TTG+TGCTT+GATCTAAC Identical SEQ ID NO: 305 SEQ ID NO: 306 miR-221 miR-221GSP9 CATGATCAGCTGGGCCAAGAGAAACCCAG miR-221RP A+GC+TACATTGTCTGC Identical overlapping sequence, SEQ ID NO: 309 SEQ ID NO: 310 ends differ miR-222 miR-222GSP8 CATGATCAGCTGGGCCAAGAGAGACCCA miR-222RP A+GC+TACATCTGGCT Identical SEQ ID NO: 311 SEQ ID NO: 312 miR-223 miR-223GSP CATGATCAGCTGGGCCAAGAGGGGTATTTG miR-223RP TG+TC+AGTTTGTCAAA Identical SEQ ID NO: 313 SEQ ID NO: 314 miR-224 miR-224GSP8 CATGATCAGCTGGGCCAAGATAAACGGA miR-224RP2 C+AAG+TCACTAGTGGTT Identical overlapping sequence, SEQ ID NO: 315 SEQ ID NO: 316 ends differ miR-296 miR-296GSP9 CATGATCAGCTGGGCCAAGAACAGGATTG miR-296RP2 A+GG+GCCCCCCCTCAA Identical SEQ ID NO: 317 SEQ ID NO: 318 miR-299 miR-299GSP9 CATGATCAGCTGGGCCAAGAATGTATGTG miR-299RP T+GG+TTTACCGTCCC Identical SEQ ID NO: 319 SEQ ID NO: 320 miR-301 miR-301GSP CATGATCAGCTGGGCCAAGAGCTTTGACAA miR-301RP C+AG+TGCAATAGTATTGT Identical SEQ ID NO: 321 SEQ ID NO: 322 miR-302a miR-302aGSP CATGATCAGCTGGGCCAAGATCACCAAAAC miR-302aRP T+AAG+TGCTTCCATGT Identical SEQ ID NO: 325 SEQ ID NO: 326 miR-320 miR-320_GSP8 CATGATCAGCTGGGCCAAGATTCGCCCT miR-320_RP3 AAAA+GCT+GGGTTGAGAGG Identical SEQ ID NO: 337 SEQ ID NO: 338 miR-323 miR-323GSP CATGATCAGCTGGGCCAAGAAGAGGTCGAC miR-323RP G+CA+CATTACACGGT Identical SEQ ID NO: 339 SEQ ID NO: 340 miR-324-3p miR-324-3pGSP CATGATCAGCTGGGCCAAGACCAGCAGCAC miR-324-3pRP C+CA+CTGCCCCAGGT Identical SEQ ID NO: 341 SEQ ID NO: 342 miR-324-5p miR-324-5pGSP CATGATCAGCTGGGCCAAGAACACCAATGC miR-324-5pRP C+GC+ATCCCCTAGGG Identical overlapping sequence, SEQ ID NO: 343 SEQ ID NO: 344 ends differ miR-325 miR-325GSP CATGATCAGCTGGGCCAAGAACACTTACTG miR-325RP C+CT+AGTAGGTGCTC one or more base pairs differ SEQ ID NO: 345 SEQ ID NO: 476 miR-326 miR-326GSP CATGATCAGCTGGGCCAAGACTGGAGGAAG miR-326RP C+CT+CTGGGCCCTTC Identical overlapping sequence, SEQ ID NO: 347 SEQ ID NO: 348 ends differ miR-328 miR-328GSP CATGATCAGCTGGGCCAAGAACGGAAGGGC miR-328RP C+TG+GCCCTCTCTGC Identical SEQ ID NO: 349 SEQ ID NO: 350 miR-330 miR-330GSP CATGATCAGCTGGGCCAAGATCTCTGCAGG miR-330RP G+CA+AAGCACAGGGC one or more base pairs differ SEQ ID NO: 351 SEQ ID NO: 478 miR-331 miR-331GSP CATGATCAGCTGGGCCAAGATTCTAGGATA miR-331RP G+CC+CCTGGGCCTAT Identical SEQ ID NO: 353 SEQ ID NO: 354 miR-337 miR-337GSP CATGATCAGCTGGGCCAAGAAAAGGCATCA miR-337RP T+TC+AGCTCCTATATG one or more base pairs differ SEQ ID NO: 355 SEQ ID NO: 490 miR-338 miR-338GSP CATGATCAGCTGGGCCAAGATCAACAAAAT miR-338RP2 T+CC+AGCATCAGTGATTT Identical SEQ ID NO: 357 SEQ ID NO: 358 miR-339 miR-339GSP9 CATGATCAGCTGGGCCAAGATGAGCTCCT miR-339RP2 T+CC+CTGTCCTCCAGG Identical SEQ ID NO: 359 SEQ ID NO: 360 miR-340 miR-340GSP CATGATCAGCTGGGCCAAGAGGCTATAAAG miR-340RP TC+CG+TCTCAGTTAC Identical SEQ ID NO: 361 SEQ ID NO: 362 miR-342 miR-342GSP3 CATGATCAGCTGGGCCAAGAGACGGGTG miR-342RP T+CT+CACACAGAAATCG Identical SEQ ID NO: 363 SEQ ID NO: 364 miR-345 miR-345GSP CATGATCAGCTGGGCCAAGAGCACTGGACT miR-345RP T+GC+TGACCCCTAGT one or more base pairs differ SEQ ID NO: 484 SEQ ID NO: 485 miR-346 miR-346GSP CATGATCAGCTGGGCCAAGAAGAGGCAGGC miR-346RP T+GT+CTGCCCGAGTG one or more base pairs differ SEQ ID NO: 367 SEQ ID NO: 488 miR-363 miR-363 GSP10 CATGATCAGCTGGGCCAAGATACAGATGGA miR-363RP AAT+TG+CAC+GGTATCC Identical SEQ ID NO: 369 SEQ ID NO: 370 miR-370 miR-370GSP CATGATCAGCTGGGCCAAGACCAGGTTCCA miR-370RP G+CC+TGCTGGGGTGG Identical overlapping sequence, SEQ ID NO: 375 SEQ ID NO: 376 ends differ miR-375 miR-375GSP CATGATCAGCTGGGCCAAGATCACGCGAGC miR-375RP TT+TG+TTCGTTCGGC Identical SEQ ID NO: 387 SEQ ID NO: 388 miR-376a miR-376aGSP3 CATGATCAGCTGGGCCAAGAACGTGGAT miR-376aRP2 A+TCGTAGA+GGAAAATCCAC one or more base pairs differ SEQ ID NO: 467 SEQ ID NO: 468 miR-378 miR-378GSP CATGATCAGCTGGGCCAAGAACACAGGACC miR-378RP C+TC+CTGACTCCAGG Identical SEQ ID NO: 391 SEQ ID NO: 392 miR-379 miR-379_GSP7 CATGATCAGCTGGGCCAAGATACGTTC miR-379RP2 T+GGT+AGACTATGGAACG Identical overlapping sequence, SEQ ID NO: 393 SEQ ID NO: 394 ends differ miR-380-5p miR-380-5pGSP CATGATCAGCTGGGCCAAGAGCGCATGTTC miR-380-5pRP T+GGT+TGACCATAGA Identical SEQ ID NO: 395 SEQ ID NO: 396 miR-380-3p miR-380-3pGSP CATGATCAGCTGGGCCAAGAAAGATGTGGA miR-380-3pRP TA+TG+TAGTATGGTCCACA one or more base pairs differ SEQ ID NO: 395 SEQ ID NO: 483 miR-381 miR-381GSP2 CATGATCAGCTGGGCCAAGAACAGAGAGC miR-381RP2 TATA+CAA+GGGCAAGCT Identical SEQ ID NO: 399 SEQ ID NO: 400 miR-382 miR-382GSP CATGATCAGCTGGGCCAAGACGAATCCACC miR-382RP G+AA+GTTGTTCGTGGT Identical SEQ ID NO: 401 SEQ ID NO: 402 miR-383 miR-383GSP CATGATCAGCTGGGCCAAGAAGCCACAGTC miR-383RP2 A+GATC+AGAAGGTGACTGT one or more base pairs differ SEQ ID NO: 465 SEQ ID NO: 466 miR-384 miR-384_GSP9 CATGATCAGCTGGGCCAAGATGTGAACAA miR-384_RP5 ATT+CCT+AG+AAATTGTTC one or more base pairs differ SEQ ID NO: 470 SEQ ID NO: 471 miR-410 miR-410 GSP9 CATGATCAGCTGGGCCAAGAACAGGCCAT miR-410RP AA+TA+TAA+CA+CAGATGGC Identical SEQ ID NO: 405 SEQ ID NO: 406 miR-412 miR-412 GSP10 CATGATCAGCTGGGCCAAGAACGGCTAGTG miR-412RP A+CTT+CACCTGGTCCACTA Identical SEQ ID NO: 407 SEQ ID NO: 408 miR-424 miR-424GSP CATGATCAGCTGGGCCAAGATCCAAAACAT miR-424RP2 C+AG+CAGCAATTCATGTTTT one or more base pairs differ SEQ ID NO: 474 SEQ ID NO: 414 miR-425 miR-425GSP CATGATCAGCTGGGCCAAGAGGCGGACACG miR-425RP A+TC+GGGAATGTCGT Identical SEQ ID NO: 417 SEQ ID NO: 418 miR-429 miR-429_GSP11 CATGATCAGCTGGGCCAAGAACGGCATTACC miR-429RP5 T+AATAC+TG+TCTGGTAATG one or more base pairs differ SEQ ID NO: 479 SEQ ID NO: 480 miR-431 miR-431 GSP10 CATGATCAGCTGGGCCAAGATGCATGACGG miR-431RP T+GT+CTTGCAGGCCG Identical overlapping sequence, SEQ ID NO: 421 SEQ ID NO: 422 ends differ miR-448 miR-448GSP CATGATCAGCTGGGCCAAGAATGGGACATC miR-448RP TTG+CATA+TGTAGGATG Identical SEQ ID NO: 423 SEQ ID NO: 424 miR-449 miR-449GSP10 CATGATCAGCTGGGCCAAGAACCAGCTAAC miR-449RP2 T+GG+CAGTGTATTGTTAGC Identical SEQ ID NO: 425 SEQ ID NO: 426 miR-450 miR-450GSP CATGATCAGCTGGGCCAAGATATTAGGAAC miR-450RP TTTT+TG+CGATGTGTT Identical SEQ ID NO: 427 SEQ ID NO: 428 miR-451 miR-451 GSP10 CATGATCAGCTGGGCCAAGAAAACTCAGTA miR-451RP AAA+CCG+TTA+CCATTACTGA Identical overlapping sequence, SEQ ID NO: 429 SEQ ID NO: 430 ends differ let7a let7a-GSP2 CATGATCAGCTGGGCCAAGAAACTATAC let7a-RP T+GA+GGTAGTAGGTTG Identical overlapping sequence, SEQ ID NO: 431 SEQ ID NO: 432 ends differ let7b let7b-GSP2 CATGATCAGCTGGGCCAAGAAACCACAC let7b-RP T+GA+GGTAGTAGGTTG Identical SEQ ID NO: 433 SEQ ID NO: 432 let7c let7c-GSP2 CATGATCAGCTGGGCCAAGAAACCATAC let7c-RP T+GA+GGTAGTAGGTTG Identical SEQ ID NO: 434 SEQ ID NO: 432 let7d let7d-GSP2 CATGATCAGCTGGGCCAAGAACTATGCA let7d-RP A+GA+GGTAGTAGGTTG Identical SEQ ID NO: 435 SEQ ID NO: 436 let7e let7e-GSP2 CATGATCAGCTGGGCCAAGAACTATACA let7e-RP T+GA+GGTAGGAGGTTG Identical SEQ ID NO: 437 SEQ ID NO: 438 let7f let7f-GSP2 CATGATCAGCTGGGCCAAGAAACTATAC let7f-RP T+GA+GGTAGTAGATTG Identical overlapping sequence, SEQ ID NO: 439 SEQ ID NO: 440 ends differ let7g let7g-GSP2 CATGATCAGCTGGGCCAAGAACTGTACA let7g-RP T+GA+GGTAGTAGTTTG Identical SEQ ID NO: 441 SEQ ID NO: 442 let7i let7i-GSP2 CATGATCAGCTGGGCCAAGAACAGCACA let7i-RP T+GA+GGTAGTAGTTTG Identical SEQ ID NO: 443 SEQ ID NO: 444

Example 5

This Example describes the detection and analysis of expression profiles for three microRNAs in total RNA isolated from twelve different tissues using methods in accordance with an embodiment of the present invention.

Methods: Quantitative analysis of miR-1, miR-124 and miR-150 microRNA templates was determined using 0.5 μg of First Choice total RNA (Ambion, Inc.) per 10 μl primer extension reaction isolated from the following tissues: brain, heart, intestine, kidney, liver, lung, lymph, ovary, skeletal muscle, spleen, thymus and uterus. The primer extension enzyme and quantitative PCR reactions were carried out as described above in EXAMPLE 3, using the following PCR primers:

miR-1 Template:

extension primer: (SEQ ID NO: 47) CATGATCAGCTGGGCCAAGATACATACTTC reverse primer: (SEQ ID NO: 48) T+G+GAA+TG+TAAAGAAGT forward primer: (SEQ ID NO: 13) CATGATCAGCTGGGCCAAGA

miR-124 Template:

extension primer: (SEQ ID NO: 149) CATGATCAGCTGGGCCAAGATGGCATTCAC reverse primer: (SEQ ID NO: 150) T+TA+AGGCACGCGGT forward primer: (SEQ ID NO: 13) CATGATCAGCTGGGCCAAGA

miR-150 template:

extension primer: (SEQ ID NO: 213) CATGATCAGCTGGGCCAAGACACTGGTA reverse primer: (SEQ ID NO: 214) T+CT+CCCAACCCTTG forward primer: (SEQ ID NO: 13) CATGATCAGCTGGGCCAAGA

Results. The expression profiles for miR-1, miR-124 and miR-150 are shown in FIGS. 3A, 3B, and 3C, respectively. The data in FIGS. 3A-3C are presented in units of microRNA copies per 10 pg of total RNA (y-axis). These units were chosen since human cell lines typically yield ≦10 pg of total RNA per cell. Hence the data shown are estimates of microRNA copies per cell. The numbers on the x-axis correspond to the following tissues: (1) brain, (2) heart, (3) intestine, (4) kidney, (5) liver, (6) lung, (7) lymph, (8) ovary, (9) skeletal muscle, (10) spleen, (11) thymus and (12) uterus.

Consistent with previous reports, very high levels of striated muscle-specific expression were found for miR-1 (as shown in FIG. 3A), and high levels of brain expression were found for miR-124 (as shown in FIG. 3B) (see Lagos-Quintana et al., RNA 9:175-179, 2003). Quantitative analysis reveals that these microRNAs are present at tens to hundreds of thousands of copies per cell. These data are in agreement with quantitative Northern blot estimates of miR-1 and miR-124 levels (see Lim et al., Nature 433:769-773, 2005). As shown in FIG. 3C, miR-150 was found to be highly expressed in the immune-related lymph node, thymus and spleen samples which is also consistent with previous findings (see Baskerville et al., RNA 11:241-247, 2005).

Example 6

This Example describes the selection and validation of primers for detecting mammalian microRNAs of interest.

Rationale: In order to perform multiple assays to detect a plurality of microRNA targets in a single sample (i.e., multiplex PCR), it is important that the assays work under uniform reverse transcriptase and PCR cycling conditions in a common buffer system with a single universal primer. The following primer design principles and high throughput assays were utilized to identify useful primer sets for desired microRNA targets that work well under the designated reaction conditions.

Primer Design:

As described in Example 2, the sensitivity of an assay to detect mammalian microRNA targets using the methods of the invention may be measured by the cycle threshold (Ct) value. The lower the Ct value (e.g., the fewer number of cycles), the more sensitive is the assay. The ΔCt value is the difference between the number of cycles (Ct) between template containing samples and no template controls, and serves as a measure of the dynamic range of the assay. Assays with a high dynamic range allow measurements of very low microRNA copy numbers. Accordingly, desirable characteristics of a microRNA detection assay include high sensitivity (low Ct value) (preferably in the range of from about 5 to about 25, such as from about 10 to about 20), and broad dynamic range (preferably in the range of from about 10 and 35, such as ΔCt≧12) between the signal of a sample containing target template and a no template background control sample.

microRNA Target Templates: Representative mammalian microRNA target templates (h=human, r=rat, m=mouse) are provided in Table 9 (SEQ ID NO:966 to SEQ ID NO:1043) which are publicly available and accessible on the World Wide Web at the Wellcome Trust Sanger Institute website in the “miRBase sequence database” as described in Griffith-Jones et al. (2004), Nucleic Acids Research 32:D109-D111 and Griffith-Jones et al. (2006), Nucleic Acids Research 34:D140-D144.

Extension Primers:

Empirical data generated as described in Examples 1-5 suggests that gene specific (GS) extension primers are primarily responsible for the dynamic range of the assays for detecting mammalian microRNA targets using the methods described herein. As described in Example 2, it was determined that the dynamic range (ΔCt) and specificity of the assays tested decreased for extension primers having gene specific regions below 6 to 7 nucleotides. Therefore, in order to optimize microRNA detection assays, extension primers were designed that have 7 to 10 nucleotide overlap with the microRNA target of interest. Exemplary extension primers for the microRNA targets listed in TABLE 9 are provided in TABLE 8 (SEQ ID NO:500 to SEQ ID NO:965). These exemplary extension primers have a gene specific (GS) region from 7 to 10 nucleotide overlap with the microRNA target of interest.

Reverse Primers:

Unmodified and locked nucleic acid (LNA)-containing reverse primers were designed to quantify the primer-extended, full length cDNA in combination with a generic universal forward primer (SEQ ID NO:13). Based on the data generated as described in Examples 1-5, it was determined that the design of the reverse primers contributes to the efficiency of the PCR reactions, with the observation that the longer the reverse primer, the better the PCR performance. However, it was also observed that the longer the overlap with the extension primer, the higher the background. Therefore, the reverse primers were designed to be as long as possible while minimizing the overlap with the gene specific portion of the extension primer, in order to reduce the non-specific background signal.

In addition, as described in Example 3, LNA base substitutions may be selected to raise the predicted Tm of the primer, with two or three LNA base substitutions typically substituted within the first 8 nucleotides from the 5′ end of the reverse primer oligonucleotide. Exemplary reverse primers for the microRNA targets listed in TABLE 9 are provided in TABLE 8. While these exemplary reverse primers contain LNA base substitutions (the “+” symbol preceding a nucleotide designates an LNA substitution), this feature is optional and not required.

Selection and validation of primers for a desired target:

Assay oligonucleotide selection is made in two steps as follows:

1) Primer designs were determined using the principles described above. Typically, 4 extension primer candidates and 2 reverse primer candidates were designed for each microRNA target of interest. The extension primers in each set overlap the gene specific region by 7, 8, 9 and 10 nucleotides, respectively, at the 3′ end. Exemplary primers designed according to these design principles are provided in TABLE 8 for the microRNA targets listed in TABLE 9.

Assay design to validate the candidate primer sets (Assay #1)

microRNA Target:

Exemplary target microRNA miR-495 has an RNA target sequence (SEQ ID NO:966) that is conserved across human (h), mouse (m) and rat (r), as indicated by the designation “hmr”-miR-495 in TABLE 9. Therefore, the primer designed for this target sequence would be expected to be useful to detect miR-495 in samples obtained from human, mouse, and rat.

microRNA miR-495 target RNA sequence: 5′ AAACAAACAUGGUGCACUUCUU 3′ (SEQ ID NO:966)

Extension Primers (4 candidates) (SEQ ID NO: 500) hmr-miR-495GS10: 5′ CATGATCAGCTGGGCCAAGAAAGAAGTGCA 3′ (SEQ ID NO: 501) hmr-miR-495GS9: 5′ CATGATCAGCTGGGCCAAGAAAGAAGTGC 3′ (SEQ ID NO: 502) hmr-miR-495GS8: 5′ CATGATCAGCTGGGCCAAGAAAGAAGTG 3′ (SEQ ID NO: 503) hmr-miR-495GS7: 5′ CATGATCAGCTGGGCCAAGAAAGAAGT Reverse Primers (2 candidates) (SEQ ID NO: 504) hmr-miR-495RP1: 5′ AAA+CAAA+CA+TGGTGCAC 3′ (SEQ ID NO: 505) hmr-miR-495RP2: 5′ AAA+C+AAA+CATGGTGC 3′

2) The primers designed as described above were tested to find pairs that showed both high sensitivity and high dynamic range in quantitative PCR assays, using the assay methods described in Example 2. The optimal combination of extension primer and reverse primer was determined for the target microRNA by testing all combinations of primers in the presence or absence of DNA template. It is preferable to use DNA rather than RNA template to test the oligo pairs because it is less likely to degrade than RNA. Degraded templates result in misleading assay data. Therefore, HPLC purified DNA template molecules are preferred.

TABLE 8 shows exemplary primer sets for use in detection assays for 78 microRNA targets (shown in TABLE 9). The candidate primers for use in these assays were designed to specifically detect human (h), mouse (m) and rat (r) microRNAs, or microRNAs from one or more species. For example, assays with the “hmr” prefix are designed to detect a perfectly conserved microRNA in all three species, whereas a “mr” prefix means the assay is designed to detect a microRNA conserved between mouse and rat, but not human. Nucleotides preceded by a plus (+) sign may be optionally locked (LNA). TABLE 9 shows the microRNA target sequence for each assay.

TABLE 8 EXEMPLARY PRIMER SETS FOR DETECTING MAMMALIAN MICRORNA TARGETS Extension Reverse Assay Number Target microRNA Primer Name Extension Primer Sequence Primer Name Reverse Primer Sequence Comments 1 hmr-miR-495 Hmr-miR- CATGATCAGCTGGGCCAAGAAAGAAGTGCA Hmr-miR- AAA+CAAA+CA+TGGTGCAC Conserved across all 495GS10 SEQ ID NO: 500 495RP1 SEQ ID NO: 504 three species Hmr-miR- CATGATCAGCTGGGCCAAGAAAGAAGTGC Hmr-miR- AAA+C+AAA+CATGGTGC 495GS9 SEQ ID NO: 501 495RP2 SEQ ID NO: 505 Hmr-miR- CATGATCAGCTGGGCCAAGAAAGAAGTG 495GS8 SEQ ID NO: 502 Hmr-miR- CATGATCAGCTGGGCCAAGAAAGAAGT 495GS7 SEQ ID NO: 503 2 mr-miR-291a- mr-mIR- CATGATCAGCTGGGCCAAGAGGCACACAAA mr-mIR-291a- AA+AG+TGCTTCCACTTTGT Mouse/rat specific; seed 3p 291a- SEQ ID NO: 506 3pRP1 SEQ ID NO: 510 region ortholog to human 3pGS10 miR-371/2 mr-mIR- CATGATCAGCTGGGCCAAGAGGCACACAA mr-mIR-291a- AA+AG+TG+CTTCCACTTT 291a-3pGS9 SEQ ID NO: 507 3pRP2 SEQ ID NO: 511 mr-mIR- CATGATCAGCTGGGCCAAGAGGCACACA 291a-3pGS8 SEQ ID NO: 508 mr-mIR- CATGATCAGCTGGGCCAAGAGGCACAC 291a-3pGS7 SEQ ID NO: 509 3 m-miR-291b- m-mIR- CATGATCAGCTGGGCCAAGAGACAAACAAA m-mIR-291b- AA+AG+TG+CAT+CCATTTTGT Mouse specific; seed 3p 291b- SEQ ID NO: 512 3pRP1 SEQ ID NO: 516 region ortholog to human 3pGS10 miR-371/2 m-mIR- CATGATCAGCTGGGCCAAGAGACAAACAA m-mIR-291b- AA+AG+TG+CATCCATTTT 291b-3pGS9 SEQ ID NO: 513 3pRP2 SEQ ID NO: 517 m-mIR- CATGATCAGCTGGGCCAAGAGACAAACA 291b-3pGS8 SEQ ID NO: 514 m-mIR- CATGATCAGCTGGGCCAAGAGACAAAC 291b-3pGS7 SEQ ID NO: 515 4 h-miR-519a h-miR- CATGATCAGCTGGGCCAAGAGTAACACTCT h-miR- AA+AG+TG+CATCCTTTTAGAGT Human specific; 519aGS10 SEQ ID NO: 518 519aRP1 SEQ ID NO: 522 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGAGTAACACTC h-miR- AA+AG+TG+CATCCTTTTAGA 519aGS9 SEQ ID NO: 519 519aRP2 SEQ ID NO: 523 h-miR- CATGATCAGCTGGGCCAAGAGTAACACT 519aGS8 SEQ ID NO: 520 h-miR- CATGATCAGCTGGGCCAAGAGTAACAC 519aGS7 SEQ ID NO: 521 5 h-miR-519b h-miR- CATGATCAGCTGGGCCAAGAAAACCTCTAA h-miR- AA+AG+TG+CATCCTTTTAG Human specific; 519bGS10 SEQ ID NO: 524 519bRP1 SEQ ID NO: 528 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGAAAACCTCTA h-miR- AA+AG+TG+CATCCTTTT 519bGS9 SEQ ID NO: 525 519bRP2 SEQ ID NO: 529 h-miR- CATGATCAGCTGGGCCAAGAAAACCTCT 519bGS8 SEQ ID NO: 526 h-miR- CATGATCAGCTGGGCCAAGAAAACCTC 519bGS7 SEQ ID NO: 527 6 h-miR-519c h-miR- CATGATCAGCTGGGCCAAGAATCCTCTAAA h-miR- AA+AG+TG+CATCTTTTTAGA Human specific; 519cGS10 SEQ ID NO: 530 519cRP1 SEQ ID NO: 534 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGAATCCTCTAA h-miR- AA+AG+TG+CATCTTTTTA 519cGS9 SEQ ID NO: 531 519cRP2 SEQ ID NO: 535 h-miR- CATGATCAGCTGGGCCAAGAATCCTCTA 519cGS8 SEQ ID NO: 532 h-miR- CATGATCAGCTGGGCCAAGAATCCTCT 519cGS7 SEQ ID NO: 533 7 h-miR-519d h-miR- CATGATCAGCTGGGCCAAGAACACTCTAAA h-miR- C+AAAG+TGCCTCCCTTTAG Human specific; 519dGS10 SEQ ID NO: 536 519dRP1 SEQ ID NO: 540 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGAACACTCTAA h-miR- C+AA+AG+TGCCTCCCTTT 519dGS9 SEQ ID NO: 537 519dRP2 SEQ ID NO: 541 h-miR- CATGATCAGCTGGGCCAAGAACACTCTA 519dGS8 SEQ ID NO: 538 h-miR- CATGATCAGCTGGGCCAAGAACACTCT 519dGS7 SEQ ID NO: 539 8 h-miR-520a h-miR- CATGATCAGCTGGGCCAAGAACAGTCCAAA h-miR- AA+AG+TGCTTCCCTTTGG Human specific; 520aGS10 SEQ ID NO: 542 520aRP1 SEQ ID NO: 546 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGAACAGTCCAA h-miR- AA+AG+T+GCTTCCCTTT 520aGS9 SEQ ID NO: 543 520aRP2 SEQ ID NO: 547 h-miR- CATGATCAGCTGGGCCAAGAACAGTCCA 520aGS8 SEQ ID NO: 544 h-miR- CATGATCAGCTGGGCCAAGAACAGTCC 520aGS7 SEQ ID NO: 545 9 h-miR-520b h-miR- CATGATCAGCTGGGCCAAGACCCTCTAAAA h-miR- AA+AG+T+GCTTCCTTTTAG Human specific; 520bGS10 SEQ ID NO: 548 520bRP1 SEQ ID NO: 552 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGACCCTCTAAA h-miR- AA+AG+TG+CTTCCTTTTA 520bGS9 SEQ ID NO: 549 520bRP2 SEQ ID NO: 553 h-miR- CATGATCAGCTGGGCCAAGACCCTCTAA 520bGS8 SEQ ID NO: 550 h-miR- CATGATCAGCTGGGCCAAGACCCTCTA 520bGS7 SEQ ID NO: 551 10 h-miR-520d h-miR- CATGATCAGCTGGGCCAAGAAACCCACCAA h-miR- AA+AG+TGCTTCTCTTTGGT Human specific; 520dGS10 SEQ ID NO: 554 520dRP1 SEQ ID NO: 558 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGAAACCCACCA h-miR- AA+AG+TG+CTTCTCTTTG 520dGS9 SEQ ID NO: 555 520dRP2 SEQ ID NO: 559 h-miR- CATGATCAGCTGGGCCAAGAAACCCACC 520dGS8 SEQ ID NO: 556 h-miR- CATGATCAGCTGGGCCAAGAAACCCAC 520dGS7 SEQ ID NO: 557 11 h-miR-520e h-miR- CATGATCAGCTGGGCCAAGACCCTCAAAAA h-miR- AA+AG+TGCTTCCTTTTTG Human specific; 520eGS10 SEQ ID NO: 560 520eRP1 SEQ ID NO: 564 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGACCCTCAAAA h-miR- AA+AG+T+GCTTCCTTTTT 520eGS9 SEQ ID NO: 561 520eRP2 SEQ ID NO: 565 h-miR- CATGATCAGCTGGGCCAAGACCCTCAAA 520eGS8 SEQ ID NO: 562 h-miR- CATGATCAGCTGGGCCAAGACCCTCAA 520eGS7 SEQ ID NO: 563 12 h-miR-520f h-miR- CATGATCAGCTGGGCCAAGAAACCCTCTAA h-miR- A+AG+TGCTTCCTTTTAGA Human specific; 520fGS10 SEQ ID NO: 566 520fRP1 SEQ ID NO: 570 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGAAACCCTCTA h-miR- A+AG+T+GCTTCCTTTTA 520fGS9 SEQ ID NO: 567 520fRP2 SEQ ID NO: 571 h-miR- CATGATCAGCTGGGCCAAGAAACCCTCT 520fGS8 SEQ ID NO: 568 h-miR- CATGATCAGCTGGGCCAAGAAACCCTC 520fGS7 SEQ ID NO: 569 13 mr-miR-329 mr-miR- CATGATCAGCTGGGCCAAGAAAAAAGGTTA mr-miR- AA+CA+CACCCAGCTAACC Specific for mouse/rat 329G510 SEQ ID NO: 572 329RP1 SEQ ID NO: 576 ortholog mr-miR- CATGATCAGCTGGGCCAAGAAAAAAGGTT mr-miR- AA+CA+CACCCAGCTAA 329GS9 SEQ ID NO: 573 329RP2 SEQ ID NO: 577 mr-miR- CATGATCAGCTGGGCCAAGAAAAAAGGT 329GS8 SEQ ID NO: 574 mr-miR- CATGATCAGCTGGGCCAAGAAAAAAGG 329GS7 SEQ ID NO: 575 14 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGAAACCCACCGA hmr-miR- AA+CATT+CATTGTTGTCGGT Conserved across all 181d 181dGS10 SEQ ID NO: 578 181dRP1 SEQ ID NO: 582 three species hmr-miR- CATGATCAGCTGGGCCAAGAAACCCACCG hmr-miR- AA+CA+TT+CATTGTTGTCG 181dGS9 SEQ ID NO: 579 181dRP2 SEQ ID NO: 583 hmr-miR- CATGATCAGCTGGGCCAAGAAACCCACC 181dGS8 SEQ ID NO: 580 hmr-miR- CATGATCAGCTGGGCCAAGAAACCCAC 181dGS7 SEQ ID NO: 581 15 has-miR-193b hmr-miR- CATGATCAGCTGGGCCAAGAAAAGCGGGAC hmr-miR- AA+CT+GGCCCTCAAAGTCCC Conserved across all 193bGS10 SEQ ID NO: 584 193bRP1 SEQ ID NO: 588 three species hmr-miR- CATGATCAGCTGGGCCAAGAAAAGCGGGA hmr-miR- AA+CT+GGCCCTCAAAGTC 193bGS9 SEQ ID NO: 585 193bRP2 SEQ ID NO: 589 hmr-miR- CATGATCAGCTGGGCCAAGAAAAGCGGG 193bGS8 SEQ ID NO: 586 hmr-miR- CATGATCAGCTGGGCCAAGAAAAGCGG 193bGS7 SEQ ID NO: 587 16 h-miR-362 h-miR- CATGATCAGCTGGGCCAAGAACTCACACCT h-miR-362RP1 AAT+CCTT+GGAACCTAGGTG Assay specific for human 362GS10 SEQ ID NO: 590 SEQ ID NO: 594 ortholog h-miR- CATGATCAGCTGGGCCAAGAACTCACACC h-miR-362RP2 AA+TC+CTT+GGAACCTAGG 362GS9 SEQ ID NO: 591 SEQ ID NO: 595 h-miR- CATGATCAGCTGGGCCAAGAACTCACAC 362GS8 SEQ ID NO: 592 h-miR- CATGATCAGCTGGGCCAAGAACTCACA 362GS7 SEQ ID NO: 593 17 mr-miR-362 mr-mIR- CATGATCAGCTGGGCCAAGATTCACACCTA mr-mIR-362- AA+TCCTT+GGAACCTAGGT Assay specific for rodent 362-3pGS10 SEQ ID NO: 596 3pRP1 SEQ ID NO: 600 ortholog mr-mIR- CATGATCAGCTGGGCCAAGATTCACACCT mr-mIR-362- AA+TC+CTT+GGAACCTAG 362-3pGS9 SEQ ID NO: 597 3pRP2 SEQ ID NO: 601 mr-mIR- CATGATCAGCTGGGCCAAGATTCACACC 362-3pGS8 SEQ ID NO: 598 mr-mIR- CATGATCAGCTGGGCCAAGATTCACAC 362-3pGS7 SEQ ID NO: 599 18 h-miR-500 h-miR- CATGATCAGCTGGGCCAAGACAGAATCCTT h-miR-500RP1 A+TG+CACCTGGGCAAGGA Assay specific for human 500GS10 SEQ ID NO: 602 SEQ ID NO: 606 ortholog h-miR- CATGATCAGCTGGGCCAAGACAGAATCCT h-miR-500RP2 A+TG+CACCTGGGCAAG 500GS9 SEQ ID NO: 603 SEQ ID NO: 607 h-miR- CATGATCAGCTGGGCCAAGACAGAATCC 500GS8 SEQ ID NO: 604 h-miR- CATGATCAGCTGGGCCAAGACAGAATC 500GS7 SEQ ID NO: 605 19 mmu-miR- mr-miR- CATGATCAGCTGGGCCAAGACTGAACCCTT mr-miR- A+TGCA+CCTGGGCAAGGG Assay specific for rodent 500 500GS10 SEQ ID NO: 608 500RP1 SEQ ID NO: 612 ortholog mr-miR- CATGATCAGCTGGGCCAAGACTGAACCCT mr-miR- A+TGCA+CCTGGGCAAG 500GS9 SEQ ID NO: 609 500RP2 SEQ ID NO: 613 mr-miR- CATGATCAGCTGGGCCAAGACTGAACCC 500GS8 SEQ ID NO: 610 mr-miR- CATGATCAGCTGGGCCAAGACTGAACC 500GS7 SEQ ID NO: 611 20 h-miR-501 h-miR- CATGATCAGCTGGGCCAAGATCTCACCCAG h-miR-501RP1 AA+T+CCTT+TGTCCCTGGG Assay specific for human 501GS10 SEQ ID NO: 614 SEQ ID NO: 618 ortholog h-miR- CATGATCAGCTGGGCCAAGATCTCACCCA h-miR-501RP2 AAT+CCTT+TGTCCCTGG 501GS9 SEQ ID NO: 615 SEQ ID NO: 619 h-miR- CATGATCAGCTGGGCCAAGATCTCACCC 501GS8 SEQ ID NO: 616 h-miR- CATGATCAGCTGGGCCAAGATCTCACC 501GS7 SEQ ID NO: 617 21 mr-miR-501 mr-miR- CATGATCAGCTGGGCCAAGATTTCACCCAG mr-miR- AA+T+CC+TTTGTCCCTGGG Assay specific for rodent 501GS10 SEQ ID NO: 620 501RP1 SEQ ID NO: 624 ortholog mr-miR- CATGATCAGCTGGGCCAAGATTTCACCCA mr-miR- AA+T+CC+TTTGTCCCTG 501GS9 SEQ ID NO: 621 501RP2 SEQ ID NO: 625 mr-miR- CATGATCAGCTGGGCCAAGATTTCACCC 501GS8 SEQ ID NO: 622 mr-miR- CATGATCAGCTGGGCCAAGATTTCACC 501GS7 SEQ ID NO: 623 22 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGAAGTGGATGAC hmr-miR- AAT+CG+TACAGGGTCAT Conserved across all 487b 487bGS10 SEQ ID NO: 626 487bRP1 SEQ ID NO: 630 three species hmr-miR- CATGATCAGCTGGGCCAAGAAGTGGATGA hmr-miR- A+AT+CG+TACAGGGTC 487bGS9 SEQ ID NO: 627 487bRP2 SEQ ID NO: 631 hmr-miR- CATGATCAGCTGGGCCAAGAAGTGGATG 487bGS8 SEQ ID NO: 628 hmr-miR- CATGATCAGCTGGGCCAAGAAGTGGAT 487bGS7 SEQ ID NO: 629 23 h-miR-489 h-miR- CATGATCAGCTGGGCCAAGAGCTGCCGTAT h-miR-489RP1 AG+TGA+CATCACATATACG Assay specific for human 489GS10 SEQ ID NO: 632 SEQ ID NO: 636 ortholog h-miR- CATGATCAGCTGGGCCAAGAGCTGCCGTA h-miR-489RP2 A+G+TGA+CATCACATATAC 489GS9 SEQ ID NO: 633 SEQ ID NO: 637 h-miR- CATGATCAGCTGGGCCAAGAGCTGCCGT 489GS8 SEQ ID NO: 634 h-miR- CATGATCAGCTGGGCCAAGAGCTGCCG 489GS7 SEQ ID NO: 635 24 m-miR-489 m-miR- CATGATCAGCTGGGCCAAGAGCTGCCATAT m-miR-489RP1 AATGA+CA+CCACATATATG Assay specific for mouse 489GS10 SEQ ID NO: 638 SEQ ID NO: 642 ortholog m-miR- CATGATCAGCTGGGCCAAGAGCTGCCATA m-miR-489RP2 AA+TGA+CA+CCACATAT 489GS9 SEQ ID NO: 639 SEQ ID NO: 643 m-miR- CATGATCAGCTGGGCCAAGAGCTGCCAT 489GS8 SEQ ID NO: 640 m-miR- CATGATCAGCTGGGCCAAGAGCTGCCA 489GS7 SEQ ID NO: 641 25 r-miR-489 r-miR- CATGATCAGCTGGGCCAAGAGCTGCCATAT r-miR-489RP1 AA+TGA+CA+TCACATATATG Assay specific for rat 489GS10 SEQ ID NO: 644 SEQ ID NO: 648 ortholog r-miR- CATGATCAGCTGGGCCAAGAGCTGCCATA r-miR-489RP2 AAT+GA+CA+TCACATATAT 489GS9 SEQ ID NO: 645 SEQ ID NO: 649 r-miR- CATGATCAGCTGGGCCAAGAGCTGCCAT 489GS8 SEQ ID NO: 646 r-miR- CATGATCAGCTGGGCCAAGAGCTGCCA 489GS7 SEQ ID NO: 647 26 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGATCAACGGGAG hmr-miR-425- AA+TGA+CACGATCACTCCC Conserved across all 425-5p 425-5pGS10 SEQ ID NO: 650 5pRP1 SEQ ID NO: 654 three species hmr-miR- CATGATCAGCTGGGCCAAGATCAACGGGA hmr-miR-425- AA+T+GA+CACGATCACTC 425-5pGS9 SEQ ID NO: 651 5pRP2 SEQ ID NO: 655 hmr-miR- CATGATCAGCTGGGCCAAGATCAACGGG 425-5pGS8 SEQ ID NO: 652 hmr-miR- CATGATCAGCTGGGCCAAGATCAACGG 425-5pGS7 SEQ ID NO: 653 27 hmr-miR-652 hmr-miR- CATGATCAGCTGGGCCAAGATGCACAACCC hmr-miR- AAT+GGCGCCACTAGGGTT Conserved across all 652GS10 SEQ ID NO: 656 652RP1 SEQ ID NO: 660 three species hmr-miR- CATGATCAGCTGGGCCAAGATGCACAACC hmr-miR- AAT+GG+CGCCACTAGGG 652GS9 SEQ ID NO: 657 652RP2 SEQ ID NO: 661 hmr-miR- CATGATCAGCTGGGCCAAGATGCACAAC 652GS8 SEQ ID NO: 658 hmr-miR- CATGATCAGCTGGGCCAAGATGCACAA 652GS7 SEQ ID NO: 659 28 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGAGAATTCATCA hmr-miR-485- AGA+GGCTGGCCGTGATG Conserved across all 485-5p 485-5pGS10 SEQ ID NO: 662 5pRP1 SEQ ID NO: 666 three species hmr-miR- CATGATCAGCTGGGCCAAGAGAATTCATC hmr-miR-485- AGA+GGCTGGCCGTGA 485-5pGS9 SEQ ID NO: 663 5pRP2 SEQ ID NO: 667 hmr-miR- CATGATCAGCTGGGCCAAGAGAATTCAT 485-5pGS8 SEQ ID NO: 664 hmr-miR- CATGATCAGCTGGGCCAAGAGAATTCA 485-5pGS7 SEQ ID NO: 665 29 has-miR-485- hmr-miR- CATGATCAGCTGGGCCAAGAAGAGAGGAGA hmr-miR-485- AG+TCATA+CACGGCTCTCC Conserved across all 3p 485-3pGS10 SEQ ID NO: 668 3pRP1 SEQ ID NO: 672 three species hmr-miR- CATGATCAGCTGGGCCAAGAAGAGAGGAG hmr-miR-485- AG+TC+ATACACGGCTCT 485-3pGS9 SEQ ID NO: 669 3pRP2 SEQ ID NO: 673 hmr-miR- CATGATCAGCTGGGCCAAGAAGAGAGGA 485-3pGS8 SEQ ID NO: 670 hmr-miR- CATGATCAGCTGGGCCAAGAAGAGAGG 485-3pGS7 SEQ ID NO: 671 30 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGACGAATATAAC hmr-miR-369- A+GA+TC+GACCGTGTTAT Conserved across all 369-5p 369-5pGS10 SEQ ID NO: 674 5pRP1 SEQ ID NO: 678 three species hmr-miR- CATGATCAGCTGGGCCAAGACGAATATAA hmr-miR-369- A+GA+TCGACCGTGTT 369-5pGS9 SEQ ID NO: 675 5pRP2 SEQ ID NO: 679 hmr-miR- CATGATCAGCTGGGCCAAGACGAATATA 369-5pGS8 SEQ ID NO: 676 hmr-miR- CATGATCAGCTGGGCCAAGACGAATAT 369-5pGS7 SEQ ID NO: 677 31 hmr-miR-671 hmr-miR- CATGATCAGCTGGGCCAAGACCTCCAGCCC hmr-miR- A+GGAAGCCCTGGAGGGGCT Conserved across all 671GS10 SEQ ID NO: 680 671RP1 SEQ ID NO: 684 three species hmr-miR- CATGATCAGCTGGGCCAAGACCTCCAGCC hmr-miR- A+GGAAGCCCTGGAGGGG 671GS9 SEQ ID NO: 681 671RP2 SEQ ID NO: 685 hmr-miR- CATGATCAGCTGGGCCAAGACCTCCAGC 671GS8 SEQ ID NO: 682 hmr-miR- CATGATCAGCTGGGCCAAGACCTCCAG 671GS7 SEQ ID NO: 683 32 h-miR-449b h-miR- CATGATCAGCTGGGCCAAGAGCCAGCTAAC h-miR- A+GGC+AGTGTATTGTTAG Assay specific for human 449bGS10 SEQ ID NO: 686 449bRP1 SEQ ID NO: 690 ortholog h-miR- CATGATCAGCTGGGCCAAGAGCCAGCTAA h-miR- AG+GC+AG+TGTATTGTT 449bGS9 SEQ ID NO: 687 449bRP2 SEQ ID NO: 691 h-miR- CATGATCAGCTGGGCCAAGAGCCAGCTA 449bGS8 SEQ ID NO: 688 h-miR- CATGATCAGCTGGGCCAAGAGCCAGCT 449bGS7 SEQ ID NO: 689 33 mr-miR-449b mr-miR- CATGATCAGCTGGGCCAAGACCAGCTAGCA mr-miR- A+GGC+AGTGCATTGCTA Assay specific for rodent 449bGS10 SEQ ID NO: 692 449bRP1 SEQ ID NO: 696 ortholog mr-miR- CATGATCAGCTGGGCCAAGACCAGCTAGC mr-miR- A+GG+CAGTGCATTGC 449bGS9 SEQ ID NO: 693 449bRP2 SEQ ID NO: 697 mr-miR- CATGATCAGCTGGGCCAAGACCAGCTAG 449bGS8 SEQ ID NO: 694 mr-miR- CATGATCAGCTGGGCCAAGACCAGCTA 449bGS7 SEQ ID NO: 695 34 m-miR-699 m-miR- CATGATCAGCTGGGCCAAGACGAGCCAGGT m-miR-699RP1 A+GGCAGTGCGACCTG Mouse specific; ortholog 699GS10 SEQ ID NO: 698 SEQ ID NO: 702 to miR-34c m-miR- CATGATCAGCTGGGCCAAGACGAGCCAGG m-miR-699RP2 A+GG+CAGTGCGACC 699GS9 SEQ ID NO: 699 SEQ ID NO: 703 m-miR- CATGATCAGCTGGGCCAAGACGAGCCAG 699GS8 SEQ ID NO: 700 m-miR- CATGATCAGCTGGGCCAAGACGAGCCA 699GS7 SEQ ID NO: 701 35 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGACAAAGTTGCT hmr-miR-409- A+GGT+TACCCGAGCAACT Conserved across all 409-5p 409-5pGS10 SEQ ID NO: 704 5pRP1 SEQ ID NO: 708 three species hmr-miR- CATGATCAGCTGGGCCAAGACAAAGTTGC hmr-miR-409- A+GG+TTACCCGAGCAA 409-5pGS9 SEQ ID NO: 705 5pRP2 SEQ ID NO: 709 hmr-miR- CATGATCAGCTGGGCCAAGACAAAGTTG 409-5pGS8 SEQ ID NO: 706 hmr-miR- CATGATCAGCTGGGCCAAGACAAAGTT 409-5pGS7 SEQ ID NO: 707 36 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGAAAGGGGTTCA hmr-miR-409- G+AA+TGTTGCTCGGTGAAC Conserved across all 409-3p 409-3pGS10 SEQ ID NO: 710 3pRP1 SEQ ID NO: 714 three species hmr-miR- CATGATCAGCTGGGCCAAGAAAGGGGTTC hmr-miR-409- G+AA+TGTTGCTCGGTGA 409-3pGS9 SEQ ID NO: 711 3pRP2 SEQ ID NO: 715 hmr-miR- CATGATCAGCTGGGCCAAGAAAGGGGTT 409-3pGS8 SEQ ID NO: 712 hmr-miR- CATGATCAGCTGGGCCAAGAAAGGGGT 409-3pGS7 SEQ ID NO: 713 37 hmr-miR-491 hmr-miR- CATGATCAGCTGGGCCAAGACCTCATGGAA hmr-miR- AG+TGG+GGAACCCTTCCA Conserved across all 491GS10 SEQ ID NO: 716 491RP1 SEQ ID NO: 720 three species hmr-miR- CATGATCAGCTGGGCCAAGACCTCATGGA hmr-miR- AG+TG+GGGAACCCTTC 491GS9 SEQ ID NO: 717 491RP2 SEQ ID NO: 721 hmr-miR- CATGATCAGCTGGGCCAAGACCTCATGG 491GS8 SEQ ID NO: 718 hmr-miR- CATGATCAGCTGGGCCAAGACCTCATG 491GS7 SEQ ID NO: 719 38 h-miR-384 h-miR- CATGATCAGCTGGGCCAAGATATGAACAAT h-miR-384RP1 A+TT+CCT+AGAAATTGTTC Assay specific for human 384GS10 SEQ ID NO: 722 SEQ ID NO: 726 ortholog h-miR- CATGATCAGCTGGGCCAAGATATGAACAA h-miR-384RP2 A+TT+CCT+AG+AAATTGT 384GS9 SEQ ID NO: 723 SEQ ID NO: 727 h-miR- CATGATCAGCTGGGCCAAGATATGAACA 384GS8 SEQ ID NO: 724 h-miR- CATGATCAGCTGGGCCAAGATATGAAC 384GS7 SEQ ID NO: 725 39 mr-miR-384 mr-miR- CATGATCAGCTGGGCCAAGATGTGAACAAT mr-miR- A+TT+CCT+AGAAATTGTT Assay specific for rodent 384GS10 SEQ ID NO: 728 384RP1 SEQ ID NO: 732 ortholog mr-miR- CATGATCAGCTGGGCCAAGATGTGAACAA mr-miR- A+TT+CCT+AG+AAATTGTT 384GS9 SEQ ID NO: 729 384RP2 SEQ ID NO: 733 mr-miR- CATGATCAGCTGGGCCAAGATGTGAACA 384GS8 SEQ ID NO: 730 mr-miR- CATGATCAGCTGGGCCAAGATGTGAAC 384GS7 SEQ ID NO: 731 40 hmr-miR-20b hmr-miR- CATGATCAGCTGGGCCAAGAACCTGCACTA hmr-miR- C+AA+AG+TGCTCATAGTGCA Conserved across all 20bGS10 SEQ ID NO: 734 20bRP1 SEQ ID NO: 738 three species hmr-miR- CATGATCAGCTGGGCCAAGAACCTGCACT hmr-miR- CAA+AG+TG+CTCATAGTG 20bGS9 SEQ ID NO: 735 20bRP2 SEQ ID NO: 739 hmr-miR- CATGATCAGCTGGGCCAAGAACCTGCAC 20bGS8 SEQ ID NO: 736 hmr-miR- CATGATCAGCTGGGCCAAGAACCTGCA 20bGS7 SEQ ID NO: 737 41 hmr-miR-490 hmr-miR- CATGATCAGCTGGGCCAAGACAGCATGGAG hmr-miR- C+AA+CCTGGAGGACTCCA Conserved across all 490GS10 SEQ ID NO: 740 490RP1 SEQ ID NO: 744 three species hmr-miR- CATGATCAGCTGGGCCAAGACAGCATGGA hmr-miR- CAA+CCT+GGAGGACTC 490GS9 SEQ ID NO: 741 490RP2 SEQ ID NO: 745 hmr-miR- CATGATCAGCTGGGCCAAGACAGCATGG 490GS8 SEQ ID NO: 742 hmr-miR- CATGATCAGCTGGGCCAAGACAGCATG 490GS7 SEQ ID NO: 743 42 hmr-miR-497 hmr-miR- CATGATCAGCTGGGCCAAGAACAAACCACA hmr-miR- C+AG+CAGCACACTGTGG Conserved across all 497GS10 SEQ ID NO: 746 497RP1 SEQ ID NO: 750 three species hmr-miR- CATGATCAGCTGGGCCAAGAACAAACCAC hmr-miR- C+AG+CAGCACACTGTG 497GS9 SEQ ID NO: 747 497RP2 SEQ ID NO: 751 hmr-miR- CATGATCAGCTGGGCCAAGAACAAACCA 497GS8 SEQ ID NO: 748 hmr-miR- CATGATCAGCTGGGCCAAGAACAAACC 497GS7 SEQ ID NO: 749 43 h-miR-301b h-miR- CATGATCAGCTGGGCCAAGATGCTTTGACA h-miR- C+AG+TG+CAATGATATTGTCA Assay specific for human 301bGS10 SEQ ID NO: 752 301bRP1 SEQ ID NO: 756 ortholog h-miR- CATGATCAGCTGGGCCAAGATGCTTTGAC h-miR- C+AG+TG+CAATGATATTGT 301bGS9 SEQ ID NO: 753 301bRP2 SEQ ID NO: 757 h-miR- CATGATCAGCTGGGCCAAGATGCTTTGA 301bGS8 SEQ ID NO: 754 h-miR- CATGATCAGCTGGGCCAAGATGCTTTG 301bGS7 SEQ ID NO: 755 44 mr-miR-301b mr-miR- CATGATCAGCTGGGCCAAGATGCTTTGACA mr-miR- C+AG+TG+CAATGGTATTGTCA Assay specific for rodent 301bGS10 SEQ ID NO: 758 301bRP1 SEQ ID NO: 762 ortholog mr-miR- CATGATCAGCTGGGCCAAGATGCTTTGAC mr-miR- C+AG+TG+CAATGGTATTGT 301bGS9 SEQ ID NO: 759 301bRP2 SEQ ID NO: 763 mr-miR- CATGATCAGCTGGGCCAAGATGCTTTGA 301bGS8 SEQ ID NO: 760 mr-miR- CATGATCAGCTGGGCCAAGATGCTTTG 301bGS7 SEQ ID NO: 761 45 hmr-miR-721 hmr-miR- CATGATCAGCTGGGCCAAGATTCCCCCTTT hmr-miR- C+AG+TG+CAATTAAAAGGG Conserved across all 721GS10 SEQ ID NO: 764 721RP1 SEQ ID NO: 768 three species hmr-miR- CATGATCAGCTGGGCCAAGATTCCCCCTT hmr-miR- C+AG+TG+CAATTAAAAG 721GS9 SEQ ID NO: 765 721RP2 SEQ ID NO: 769 hmr-miR- CATGATCAGCTGGGCCAAGATTCCCCCT 721GS8 SEQ ID NO: 766 hmr-miR- CATGATCAGCTGGGCCAAGATTCCCCC 721GS7 SEQ ID NO: 767 46 hmr-miR-532 hmr-miR- CATGATCAGCTGGGCCAAGAACGGTCCTAC hmr-miR- CA+TG+CCTTGAGTGTAGG Conserved across all 532GS10 SEQ ID NO: 770 532RP1 SEQ ID NO: 774 three species hmr-miR- CATGATCAGCTGGGCCAAGAACGGTCCTA hmr-miR- CA+TG+CCTTGAGTGTA 532GS9 SEQ ID NO: 771 532RP2 SEQ ID NO: 775 hmr-miR- CATGATCAGCTGGGCCAAGAACGGTCCT 532GS8 SEQ ID NO: 772 hmr-miR- CATGATCAGCTGGGCCAAGAACGGTCC 532GS7 SEQ ID NO: 773 47 h-miR-488 h-miR- CATGATCAGCTGGGCCAAGATTGAGAGTGC h-miR-488RP1 C+CCA+GATAATGGCACT Assay specific for human 488GS10 SEQ ID NO: 776 SEQ ID NO: 780 ortholog h-miR- CATGATCAGCTGGGCCAAGATTGAGAGTG h-miR-488RP2 C+CC+A+GATAATGGCA 488GS9 SEQ ID NO: 777 SEQ ID NO: 781 h-miR- CATGATCAGCTGGGCCAAGATTGAGAGT 488GS8 SEQ ID NO: 778 h-miR- CATGATCAGCTGGGCCAAGATTGAGAG 488GS7 SEQ ID NO: 779 48 mr-miR-488 mr-miR- CATGATCAGCTGGGCCAAGATTGAGAGTGC mr-miR- C+CCA+GATAATAGCACT Assay specific for rodent 488GS10 SEQ ID NO: 782 488RP1 SEQ ID NO: 786 ortholog mr-miR- CATGATCAGCTGGGCCAAGATTGAGAGTG mr-miR- C+CC+A+GATAATAGCA 488GS9 SEQ ID NO: 783 488RP2 SEQ ID NO: 787 mr-miR- CATGATCAGCTGGGCCAAGATTGAGAGT 488GS8 SEQ ID NO: 784 mr-miR- CATGATCAGCTGGGCCAAGATTGAGAG 488GS7 SEQ ID NO: 785 49 hmr-miR-539 hmr-miR- CATGATCAGCTGGGCCAAGAACACACCAAG hmr-miR- GG+AG+AAATTATCCTTGGT Conserved across all 539GS10 SEQ ID NO: 788 539RP1 SEQ ID NO: 792 three species hmr-miR- CATGATCAGCTGGGCCAAGAACACACCAA hmr-miR- G+GA+G+AAATTATCCTTGG 539GS9 SEQ ID NO: 789 539RP2 SEQ ID NO: 793 hmr-miR- CATGATCAGCTGGGCCAAGAACACACCA 539GS8 SEQ ID NO: 790 hmr-miR- CATGATCAGCTGGGCCAAGAACACACC 539GS7 SEQ ID NO: 791 50 h-miR-505 h-miR- CATGATCAGCTGGGCCAAGAGAGGAAACCA h-miR-505RP1 GT+CAA+CACTTGCTGGTT Assay specific for human 505GS10 SEQ ID NO: 794 SEQ ID NO: 798 ortholog h-miR- CATGATCAGCTGGGCCAAGAGAGGAAACC h-miR-505RP2 G+T+CAA+CACTTGCTGG 505GS9 SEQ ID NO: 795 SEQ ID NO: 799 h-miR- CATGATCAGCTGGGCCAAGAGAGGAAAC 505GS8 SEQ ID NO: 796 h-miR- CATGATCAGCTGGGCCAAGAGAGGAAA 505GS7 SEQ ID NO: 797 51 mr-miR-505 mr-miR- CATGATCAGCTGGGCCAAGAGGAAACCAGC mr-miR- CG+T+CAA+CA+CTTGCTGGT Assay specific for rodent 505GS10 SEQ ID NO: 800 505RP1 SEQ ID NO: 804 ortholog mr-miR- CATGATCAGCTGGGCCAAGAGGAAACCAG mr-miR- CG+T+CAA+CA+CTTGCTG 505GS9 SEQ ID NO: 801 505RP2 SEQ ID NO: 805 mr-miR- CATGATCAGCTGGGCCAAGAGGAAACCA 505GS8 SEQ ID NO: 802 mr-miR- CATGATCAGCTGGGCCAAGAGGAAACC 505GS7 SEQ ID NO: 803 52 h-miR-18b h-miR- CATGATCAGCTGGGCCAAGATAACTGCACT h-miR-18bRP1 TAA+GG+TGCATCTAGTGC Assay specific for human 18bGS10 SEQ ID NO: 806 SEQ ID NO: 810 ortholog h-miR- CATGATCAGCTGGGCCAAGATAACTGCAC h-miR-18bRP2 T+AA+GG+TGCATCTAGT 18bGS9 SEQ ID NO: 807 SEQ ID NO: 811 h-miR- CATGATCAGCTGGGCCAAGATAACTGCA 18bGS8 SEQ ID NO: 808 h-miR- CATGATCAGCTGGGCCAAGATAACTGC 18bGS7 SEQ ID NO: 809 53 mr-miR-18b mr-miR- CATGATCAGCTGGGCCAAGATAACAGCACT mr-miR- T+AA+GG+TGCATCTAGTGC Assay specific for rodent 18bGS10 SEQ ID NO: 812 18bRP1 SEQ ID NO: 816 ortholog mr-miR- CATGATCAGCTGGGCCAAGATAACAGCAC mr-miR- TAA+GG+TG+CATCTAGT 18bGS9 SEQ ID NO: 813 18bRP2 SEQ ID NO: 817 mr-miR- CATGATCAGCTGGGCCAAGATAACAGCA 18bGS8 SEQ ID NO: 814 mr-miR- CATGATCAGCTGGGCCAAGATAACAGC 18bGS7 SEQ ID NO: 815 54 hmr-miR-503 hmr-miR- CATGATCAGCTGGGCCAAGACAGTACTGTT hmr-miR- T+AGC+AGCGGGAACAGT Conserved across all 503GS10 SEQ ID NO: 818 503RP1 SEQ ID NO: 822 three species hmr-miR- CATGATCAGCTGGGCCAAGACAGTACTGT hmr-miR- T+AGC+AGCGGGAACA 503GS9 SEQ ID NO: 819 503RP2 SEQ ID NO: 823 hmr-miR- CATGATCAGCTGGGCCAAGACAGTACTG 503GS8 SEQ ID NO: 820 hmr-miR- CATGATCAGCTGGGCCAAGACAGTACT 503GS7 SEQ ID NO: 821 55 hmr-miR-455 hmr-miR- CATGATCAGCTGGGCCAAGACGATGTAGTC hmr-miR- TA+TG+TGCCTTTGGACTA Conserved across all 455GS10 SEQ ID NO: 824 455RP1 SEQ ID NO: 828 three species hmr-miR- CATGATCAGCTGGGCCAAGACGATGTAGT hmr-miR- TA+TG+TGCCTTTGGAC 455GS9 SEQ ID NO: 825 455RP2 SEQ ID NO: 829 hmr-miR- CATGATCAGCTGGGCCAAGACGATGTAG 455GS8 SEQ ID NO: 826 hmr-miR- CATGATCAGCTGGGCCAAGACGATGTA 455GS7 SEQ ID NO: 827 56 hmr-miR-92b hmr-miR- CATGATCAGCTGGGCCAAGAGAGGCCGGGA hmr-miR- TAT+TG+CACTCGTCCCG Conserved across all 92bGS10 SEQ ID NO: 830 92bRP1 SEQ ID NO: 834 three species hmr-miR- CATGATCAGCTGGGCCAAGAGAGGCCGGG hmr-miR- TAT+TG+CACTCGTCCC 92bGS9 SEQ ID NO: 831 92bRP2 SEQ ID NO: 835 hmr-miR- CATGATCAGCTGGGCCAAGAGAGGCCGG 92bGS8 SEQ ID NO: 832 hmr-miR- CATGATCAGCTGGGCCAAGAGAGGCCG 92bGS7 SEQ ID NO: 833 57 h-miR-483 h-miR- CATGATCAGCTGGGCCAAGAAGAAGACGGG h-miR-483RP1 T+CAC+TCCTCTCCTCCCGT Assay specific for human 483GS10 SEQ ID NO: 836 SEQ ID NO: 840 ortholog h-miR- CATGATCAGCTGGGCCAAGAAGAAGACGG h-miR-483RP2 T+CAC+TCCTCTCCTCCC 483GS9 SEQ ID NO: 837 SEQ ID NO: 841 h-miR- CATGATCAGCTGGGCCAAGAAGAAGACG 483GS8 SEQ ID NO: 838 h-miR- CATGATCAGCTGGGCCAAGAAGAAGAC 483GS7 SEQ ID NO: 839 58 mr-miR-483 mr-miR- CATGATCAGCTGGGCCAAGAACAAGACGGG mr-miR- TC+ACTCCTCCCCTCCCGT Assay specific for rodent 483GS10 SEQ ID NO: 842 483RP1 SEQ ID NO: 846 ortholog mr-miR- CATGATCAGCTGGGCCAAGAACAAGACGG mr-miR- TC+ACTCCTCCCCTCCC 483GS9 SEQ ID NO: 843 483RP2 SEQ ID NO: 847 mr-miR- CATGATCAGCTGGGCCAAGAACAAGACG 483GS8 SEQ ID NO: 844 mr-miR- CATGATCAGCTGGGCCAAGAACAAGAC 483GS7 SEQ ID NO: 845 59 hmr-miR-484 hmr-miR- CATGATCAGCTGGGCCAAGAATCGGGAGGG hmr-miR- TCA+GGCTCAGTCCCCTC Conserved across all 484GS10 SEQ ID NO: 848 484RP1 SEQ ID NO: 852 three species hmr-miR- CATGATCAGCTGGGCCAAGAATCGGGAGG hmr-miR- TC+AGGCTCAGTCCCC 484GS9 SEQ ID NO: 849 484RP2 SEQ ID NO: 853 hmr-miR- CATGATCAGCTGGGCCAAGAATCGGGAG 484GS8 SEQ ID NO: 850 hmr-miR- CATGATCAGCTGGGCCAAGAATCGGGA 484GS7 SEQ ID NO: 851 60 mmu-miR- hmr-miR- CATGATCAGCTGGGCCAAGACAGGCTCAAA hmr-miR- TC+CCTGAGGAGCCCTTTGA Rodent specific; ortholog 351 351GS10 SEQ ID NO: 854 351RP1 SEQ ID NO: 858 to human miR-125 hmr-miR- CATGATCAGCTGGGCCAAGACAGGCTCAA hmr-miR- TC+CCTGAGGAGCCCTTT 351GS9 SEQ ID NO: 855 351RP2 SEQ ID NO: 859 hmr-miR- CATGATCAGCTGGGCCAAGACAGGCTCA 351GS8 SEQ ID NO: 856 hmr-miR- CATGATCAGCTGGGCCAAGACAGGCTC 351GS7 SEQ ID NO: 857 61 hmr-miR-615 hmr-miR- CATGATCAGCTGGGCCAAGAAGAGGGAGAC hmr-miR- TC+CGAGCCTGGGTCTC Conserved across all 615GS10 SEQ ID NO: 860 615RP1 SEQ ID NO: 864 three species hmr-miR- CATGATCAGCTGGGCCAAGAAGAGGGAGA hmr-miR- TC+CGAGCCTGGGTC 615GS9 SEQ ID NO: 861 615RP2 SEQ ID NO: 865 hmr-miR- CATGATCAGCTGGGCCAAGAAGAGGGAG 615GS8 SEQ ID NO: 862 hmr-miR- CATGATCAGCTGGGCCAAGAAGAGGGA 615GS7 SEQ ID NO: 863 62 hmr-miR-486 hmr-miR- CATGATCAGCTGGGCCAAGACTCGGGGCAG hmr-miR- T+CC+TGTACTGAGCTGCC Conserved across all 486GS10 SEQ ID NO: 866 486RP1 SEQ ID NO: 870 three species hmr-miR- CATGATCAGCTGGGCCAAGACTCGGGGCA hmr-miR- T+CC+TGTACTGAGCTG 486GS9 SEQ ID NO: 867 486RP2 SEQ ID NO: 871 hmr-miR- CATGATCAGCTGGGCCAAGACTCGGGGC 486GS8 SEQ ID NO: 868 hmr-miR- CATGATCAGCTGGGCCAAGACTCGGGG 486GS7 SEQ ID NO: 869 63 hmr-miR-494 hmr-miR- CATGATCAGCTGGGCCAAGAAGGTTTCCCG hmr-miR- T+GA+AA+CATACACGGGA Conserved across all 494GS10 SEQ ID NO: 872 494RP1 SEQ ID NO: 876 three species hmr-miR- CATGATCAGCTGGGCCAAGAAGGTTTCCC hmr-miR- T+GA+AA+CATACACGG 494GS9 SEQ ID NO: 873 494RP2 SEQ ID NO: 877 hmr-miR- CATGATCAGCTGGGCCAAGAAGGTTTCC 494GS8 SEQ ID NO: 874 hmr-miR- CATGATCAGCTGGGCCAAGAAGGTTTC 494GS7 SEQ ID NO: 875 64 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGACTGGCACACA hmr-miR-493- T+GAA+GGTCTACTGTG Conserved across all 493-3p 493-3pGS10 SEQ ID NO: 878 3pRP1 SEQ ID NO: 882 three species hmr-miR- CATGATCAGCTGGGCCAAGACTGGCACAC hmr-miR-493- T+GAA+GGTCTACTGT 493-3pGS9 SEQ ID NO: 879 3pRP2 SEQ ID NO: 883 hmr-miR- CATGATCAGCTGGGCCAAGACTGGCACA 493-3pGS8 SEQ ID NO: 880 hmr-miR- CATGATCAGCTGGGCCAAGACTGGCAC 493-3pGS7 SEQ ID NO: 881 65 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGAAGCCTATGGA hmr-miR- T+GA+GAAC+TGAATTCCATA Conserved across all 146b 146bGS10 SEQ ID NO: 884 146bRP1 SEQ ID NO: 888 three species hmr-miR- CATGATCAGCTGGGCCAAGAAGCCTATGG hmr-miR- T+GA+GAAC+TGAATTCCA 146bGS9 SEQ ID NO: 885 146bRP2 SEQ ID NO: 889 hmr-miR- CATGATCAGCTGGGCCAAGAAGCCTATG 146bGS8 SEQ ID NO: 886 hmr-miR- CATGATCAGCTGGGCCAAGAAGCCTAT 146bGS7 SEQ ID NO: 887 66 r-miR-1 r-miR- CATGATCAGCTGGGCCAAGATACACACTTC r-miR-1RP1 T+G+GAA+TGTAAAGAAGTG Assay specific for rat 1GS10 SEQ ID NO: 890 SEQ ID NO: 894 ortholog r-miR-1GS9 CATGATCAGCTGGGCCAAGATACACACTT r-miR-1RP2 T+G+GAA+TGTAAAGAAG SEQ ID NO: 891 SEQ ID NO: 895 r-miR-1GS8 CATGATCAGCTGGGCCAAGATACACACT SEQ ID NO: 892 r-miR-1GS7 CATGATCAGCTGGGCCAAGATACACAC SEQ ID NO: 893 67 h-miR-675-5p h-miR-675- CATGATCAGCTGGGCCAAGACACTGTGGGC h-miR-675- T+GGTGCGGAGAGGGCCCA Assay specific for human 5pGS10 SEQ ID NO: 896 5pRP1 SEQ ID NO: 900 ortholog h-miR-675- CATGATCAGCTGGGCCAAGACACTGTGGG h-miR-675- T+GGTGCGGAGAGGGC 5pGS9 SEQ ID NO: 897 5pRP2 SEQ ID NO: 901 h-miR-675- CATGATCAGCTGGGCCAAGACACTGTGG 5pGS8 SEQ ID NO: 898 h-miR-675- CATGATCAGCTGGGCCAAGACACTGTG 5pGS7 SEQ ID NO: 899 68 mr-miR-675- mr-miR- CATGATCAGCTGGGCCAAGAACTGTGGGCC mr-miR-675- T+GGTGCGGAAAGGGCC Assay specific for rodent 5p 675-5pGS10 SEQ ID NO: 902 5pRP1 SEQ ID NO: 906 ortholog mr-miR- CATGATCAGCTGGGCCAAGAACTGTGGGC mr-miR-675- T+GGTGCGGAAAGGG 675-5pGS9 SEQ ID NO: 903 5pRP2 SEQ ID NO: 907 mr-miR- CATGATCAGCTGGGCCAAGAACTGTGGG 675-5pGS8 SEQ ID NO: 904 mr-miR- CATGATCAGCTGGGCCAAGAACTGTGG 675-5pGS7 SEQ ID NO: 905 69 hmr-miR-668 hmr-miR- CATGATCAGCTGGGCCAAGAGTAGTGGGCC hmr-miR- TG+TCACTCGGCTCGGCC Conserved across all 668GS10 SEQ ID NO: 908 668RP1 SEQ ID NO: 912 three species hmr-miR- CATGATCAGCTGGGCCAAGAGTAGTGGGC hmr-miR- TG+TCACTCGGCTCGG 668GS9 SEQ ID NO: 909 668RP2 SEQ ID NO: 913 hmr-miR- CATGATCAGCTGGGCCAAGAGTAGTGGG 668GS8 SEQ ID NO: 910 hmr-miR- CATGATCAGCTGGGCCAAGAGTAGTGG 668GS7 SEQ ID NO: 911 70 r-miR-346 r-miR- CATGATCAGCTGGGCCAAGAAGAGGCAGGC r-miR-346RP1 TGTC+TGCCTGAGTGCCTG Assay specific for rat 346GS10 SEQ ID NO: 914 SEQ ID NO: 918 ortholog r-miR- CATGATCAGCTGGGCCAAGAAGAGGCAGG r-miR-346RP2 TGTC+TGCCTGAGTGCC 346GS9 SEQ ID NO: 915 SEQ ID NO: 919 r-miR- CATGATCAGCTGGGCCAAGAAGAGGCAG 346GS8 SEQ ID NO: 916 r-miR- CATGATCAGCTGGGCCAAGAAGAGGCA 346GS7 SEQ ID NO: 917 71 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGATTCAGTTATC hmr-miR-542- TG+TGA+CAGATTGATAACT Conserved across all 542-3p 542-3pGS10 SEQ ID NO: 920 3pRP1 SEQ ID NO: 924 three species hmr-miR- CATGATCAGCTGGGCCAAGATTCAGTTAT hmr-miR-542- TG+T+GA+CAGATTGATAA 542-3pGS9 SEQ ID NO: 921 3pRP2 SEQ ID NO: 925 hmr-miR- CATGATCAGCTGGGCCAAGATTCAGTTA 542-3pGS8 SEQ ID NO: 922 hmr-miR- CATGATCAGCTGGGCCAAGATTCAGTT 542-3pGS7 SEQ ID NO: 923 72 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGACGTGACATGATG hmr-miR-542- CTC+GG+GGATCATCATG Conserved across all 542-5p 542-5pGS10 SEQ ID NO: 926 5pRP1 SEQ ID NO: 930 three species hmr-miR- CATGATCAGCTGGGCCAAGACGTGACATG hmr-miR-542- C+TC+GGGGATCATCAT 542-5pGS9 SEQ ID NO: 927 5pRP2 SEQ ID NO: 931 hmr-miR- CATGATCAGCTGGGCCAAGACGTGACAT 542-5pGS8 SEQ ID NO: 928 hmr-miR- CATGATCAGCTGGGCCAAGACGTGACA 542-5pGS7 SEQ ID NO: 929 73 hmr-miR-499 hmr-miR- CATGATCAGCTGGGCCAAGAAAACATCACT hmr-miR- T+TAA+GA+CTTGCAGTGAT Conserved across all 499G510 SEQ ID NO: 932 499RP1 SEQ ID NO: 936 three species hmr-miR- CATGATCAGCTGGGCCAAGAAAACATCAC hmr-miR- T+TAA+GA+CTTGCAGTG 499GS9 SEQ ID NO: 933 499RP2 SEQ ID NO: 937 hmr-miR- CATGATCAGCTGGGCCAAGAAAACATCA 499GS8 SEQ ID NO: 934 hmr-miR- CATGATCAGCTGGGCCAAGAAAACATC 499GS7 SEQ ID NO: 935 74 hmr-miR-758 hmr-miR- CATGATCAGCTGGGCCAAGAGTTAGTGGAC hmr-miR- TT+TG+TGACCTGGTCCAC Conserved across all 758GS10 SEQ ID NO: 938 758RP1 SEQ ID NO: 942 three species hmr-miR- CATGATCAGCTGGGCCAAGAGTTAGTGGA hmr-miR- TT+TG+T+GACCTGGTCC 758GS9 SEQ ID NO: 939 758RP2 SEQ ID NO: 943 hmr-miR- CATGATCAGCTGGGCCAAGAGTTAGTGG 758GS8 SEQ ID NO: 940 hmr-miR- CATGATCAGCTGGGCCAAGAGTTAGTG 758GS7 SEQ ID NO: 941 75 hmr-miR-194 miR- CATGATCAGCTGGGCCAAGATCCACATGGA miR-194RP1 TG+TAA+CAGCAACTCCA Conserved across all 194GSP10 SEQ ID NO: 944 SEQ ID NO: 948 three species miR- CATGATCAGCTGGGCCAAGATCCACATGG miR-RP2 TG+TAA+CA+GCAACTCCAT 194GSP9 SEQ ID NO: 945 SEQ ID NO: 949 miR- CATGATCAGCTGGGCCAAGATCCACATG 194GSP8 SEQ ID NO: 946 miR- CATGATCAGCTGGGCCAAGATCCACAT 194GSP7 SEQ ID NO: 947 76 hmr-miR-206 mir- CATGATCAGCTGGGCCAAGACCACACACTT mir-206RP1 T+GGAA+TGTAAGGAAGT Conserved across all 206GSP10 SEQ ID NO: 950 SEQ ID NO: 954 three species mir- CATGATCAGCTGGGCCAAGACCACACACT miR-206RP2 T+G+GAA+TGTAAGGAAGTGT 206GSP9 SEQ ID NO: 951 SEQ ID NO: 955 mir- CATGATCAGCTGGGCCAAGACCACACAC 206GSP8 SEQ ID NO: 952 mir- CATGATCAGCTGGGCCAAGACCACACA 206GSP7 SEQ ID NO: 953 77 hmr-miR-1 miR-1GS10 CATGATCAGCTGGGCCAAGATACATACTTC miR-1RP1 TG+GAA+TG+TAAAGAAGTA Conserved across all (SEQ ID NO: 47) (SEQ ID NO: 959) three species miR-1GS9 CATGATCAGCTGGGCCAAGATACATACTT (SEQ miR-1RP2 T+G+GAA+TG+TAAAGAAGT ID NO: 956) (SEQ ID NO: 48) miR-1GS8 CATGATCAGCTGGGCCAAGATACATACT (SEQ ID NO: 957) miR-1GS7 CATGATCAGCTGGGCCAAGATACATAC (SEQ ID NO: 958) 78 hmr-miR-9 miR-9GS10 CATGATCAGCTGGGCCAAGATCATACAGCT miR-9RP1 T+CTTT+GGTTATCTAGCT (SEQ Conserved across all (SEQ ID NO: 960) ID NO: 964) three species miR-9G59 CATGATCAGCTGGGCCAAGATCATACAGC (SEQ miR-9RP2 TC+TTT+GGTT+ATCTAGCTGTA ID NO: 961) (SEQ ID NO: 965) miR-9G58 CATGATCAGCTGGGCCAAGATCATACAG (SEQ ID NO: 962) miR-9G57 CATGATCAGCTGGGCCAAGATCATACA (SEQ ID NO: 963)

TABLE 9 SEQ Assay Target ID Number MicroRNA Name RNA target sequence NO: 1. hmr-miR-495 AAACAAACAUGGUGCACUUCUU 966 2. mr-miR-291a- AAAGUGCUUCCACUUUGUGUGCC 967 3p 3. m-mIR-291b-3p AAAGUGCAUCCAUUUUGUUUGUC 968 4. h-miR-519a AAAGUGCAUCCUUUUAGAGUGUUAC 969 5. h-miR-519b AAAGUGCAUCCUUUUAGAGGUUU 970 6. h-miR-519c AAAGUGCAUCUUUUUAGAGGAU 971 7. h-miR-519d CAAAGUGCCUCCCUUUAGAGUGU 972 8. h-miR-520a AAAGUGCUUCCCUUUGGACUGU 973 9. h-miR-520b AAAGUGCUUCCUUUUAGAGGG 974 10. h-miR-520d AAAGUGCUUCUCUUUGGUGGGUU 975 11. h-miR-520e AAAGUGCUUCCUUUUUGAGGG 976 12. h-miR-520f AAGUGCUUCCUUUUAGAGGGUU 977 13. mr-miR-329 AACACACCCAGCUAACCUUUUU 978 14. hmr-miR-181d AACAUUCAUUGUUGUCGGUGGGUU 979 15. hmr-miR-193b AACUGGCCCUCAAAGUCCCGCUUU 980 16. h-miR-362 AAUCCUUGGAACCUAGGUGUGAGU 981 17. mr-mIR-362-3p AAUCCUUGGAACCUAGGUGUGAA 982 18. h-miR-500 AUGCACCUGGGCAAGGAUUCUG 983 19. mr-miR-500 AUGCACCUGGGCAAGGGUUCAG 984 20. h-miR-501 AAUCCUUUGUCCCUGGGUGAGA 985 21. mr-miR-501 AAUCCUUUGUCCCUGGGUGAAA 986 22. hmr-miR-487b AAUCGUACAGGGUCAUCCACU 987 23. h-miR-489 AGUGACAUCACAUAUACGGCAGC 988 24. m-miR-489 AAUGACACCACAUAUAUGGCAGC 989 25. r-miR-489 AAUGACAUCACAUAUAUGGCAGC 990 26. hmr-miR-425- AAUGACACGAUCACUCCCGUUGA 991 5p 27. hmr-miR-652 AAUGGCGCCACUAGGGUUGUGCA 992 28. hmr-miR-485 AGAGGCUGGCCGUGAUGAAUUC 993 -5p 29. hmr-miR-485 AGUCAUACACGGCUCUCCUCUCU 994 -3p 30. hmr-miR-369 AGAUCGACCGUGUUAUAUUCG 995 -5p 31. hmr-miR-671 AGGAAGCCCUGGAGGGGCUGGAGG 996 32. h-miR-449b AGGCAGUGUAUUGUUAGCUGGC 997 33. mr-miR-449b AGGCAGUGCAUUGCUAGCUGG 998 34. m-miR-699 AGGCAGUGCGACCUGGCUCG 999 35. hmr-miR-409- AGGUUACCCGAGCAACUUUGCA 1000 5p 36. hmr-miR-409- GAAUGUUGCUCGGUGAACCCCUU 1001 3p 37. hmr-miR-491 AGUGGGGAACCCUUCCAUGAGG 1002 38. h-miR-384 AUUCCUAGAAAUUGUUCAUA 1003 39. mr-miR-384 AUUCCUAGAAAUUGUUCACA 1004 40. hmr-miR-20b CAAAGUGCUCAUAGUGCAGGUAG 1005 41. hmr-miR-490 CAACCUGGAGGACUCCAUGCUG 1006 42. hmr-miR-497 CAGCAGCACACUGUGGUUUGU 1007 43. h-miR-301b CAGUGCAAUGAUAUUGUCAAAGCA 1008 44. mr-miR-301b CAGUGCAAUGGUAUUGUCAAAGCA 1009 45. hmr-miR-721 CAGUGCAAUUAAAAGGGGGAA 1010 46. hmr-miR-532 CAUGCCUUGAGUGUAGGACCGU 1011 47. h-miR-488 CCCAGAUAAUGGCACUCUCAA 1012 48. mr-miR-488 CCCAGAUAAUAGCACUCUCAA 1013 49. hmr-miR-539 GGAGAAAUUAUCCUUGGUGUGU 1014 50. h-miR-505 GUCAACACUUGCUGGUUUCCUC 1015 51. mr-miR-505 CGUCAACACUUGCUGGUUUUCU 1016 52. h-miR-18b UAAGGUGCAUCUAGUGCAGUUA 1017 53. mr-miR-18b UAAGGUGCAUCUAGUGCUGUUA 1018 54. hmr-miR-503 UAGCAGCGGGAACAGUACUGC 1019 55. hmr-miR-455 UAUGUGCCUUUGGACUACAUCG 1020 56. hmr-miR-92b UAUUGCACUCGUCCCGGCCUC 1021 57. h-miR-483 UCACUCCUCUCCUCCCGUCUUCU 1022 58. mr-miR-483 UCACUCCUCCCCUCCCGUCUUGU 1023 59. hmr-miR-484 UCAGGCUCAGUCCCCUCCCGAU 1024 60. hmr-miR-351 UCCCUGAGGAGCCCUUUGAGCCUG 1025 61. hmr-miR-615 UCCGAGCCUGGGUCUCCCUCU 1026 62. hmr-miR-486 UCCUGUACUGAGCUGCCCCGAG 1027 63. hmr-miR-494 UGAAACAUACACGGGAAACCU 1028 64. hmr-miR-493- UGAAGGUCUACUGUGUGCCAG 1029 3p 65. hmr-miR-146b UGAGAACUGAAUUCCAUAGGCU 1030 66. r-miR-1 UGGAAUGUAAAGAAGUGUGUA 1031 67. h-miR-675-5p UGGUGCGGAGAGGGCCCACAGUG 1032 68. mr-miR-675-5p UGGUGCGGAAAGGGCCCACAGU 1033 69. hmr-miR-668 UGUCACUCGGCUCGGCCCACUAC 1034 70. r-miR-346 UGUCUGCCUGAGUGCCUGCCUCU 1035 71. hmr-miR-542- UGUGACAGAUUGAUAACUGAAA 1036 3p 72. hmr-miR-542- CUCGGGGAUCAUCAUGUCACG 1037 5p 73. hmr-miR-499 UUAAGACUUGCAGUGAUGUUU 1038 74. hmr-miR-758 UUUGUGACCUGGUCCACUAACC 1039 75. hmiR-194 UGUAACAGCAACUCCAUGUGGA 1040 76. hmiR-206 UGGAAUGUAAGGAAGUGUGUGG 1041 77. hmiR-1 UGGAAUGUAAAGAAGUAUGUA 1042 78. hmiR-9 UCUUUGGUUAUCUAGCUGUAUGA 1043

Assay Format:

Several candidate primer sets shown above in TABLE 8 were tested in a high-throughput assay testing format as follows:

Each test assay (e.g., assay #75, #76, #77 and #78 listed in TABLE 8) was run in 4×4 wells of a 96 well plate, with 6 assays per 96 well plate, thereby allowing for rapid determination of the optimal primer pair for each target.

For each assay, each of the 4 candidate extension (GS) primers were tested in a separate row of the 96 well plate. Each of the 2 reverse primers were tested plus (1 nM DNA) or minus template (10 mM Tris pH 7.6, 0.1 mM EDTA, 100 ng/ul yeast total RNA).

Following reverse transcription, one set of duplicate non-template control and template samples was tested against reverse primer 1 (RP1) and the other against reverse primer 2 (RP2).

Reverse Transcriptase Assay Conditions:

    • 6 μl of RT master mix was added to all 96 wells
    • 2 μl of 0.5 μM GS primers was added to four successive wells
    • yeast RNA in TE (10 mM Tris pH 7.6, 0.1 mM EDTA) was added to all odd-numbered wells and pre-diluted DNA templates was added to even-numbered wells

Samples were mixed well and the reverse transcriptase step was carried out, followed by dilution with 80 μl TE (10 mM Tris pH 7.6, 0.1 mM EDTA).

2 μl of the reverse transcription mixture was transferred into quadruplicate wells of a 384 well PCR plate preloaded with 80 PCR mix per well containing universal primer plus the appropriate reverse primers.

The quantitative PCR reaction results were evaluated on a real-time PCR instrument compatible with 384 well plates.

Ct values for the PCR reactions were determined based on a baseline threshold of 0.01. The sensitivity (Ct value of 1 nM template) and dynamic range (Ct of no-template control minus the Ct of the 1 nM template) were determined for each primer pair in each assay. The results of exemplary assays #75, #76, #77 and #78, listed in TABLE 8, are shown in TABLE 10 below.

TABLE 10 ASSAY RESULTS USING CANDIDATE PRIMER SETS FOR DETECTING MIR-1, MIR-9; MIR-194 AND MIR-206 Selected microRNA Dynamic for use in target Extension primer Reverse primer Sensitivity Range profiling miR-9 miR-9GS10 miR-9 RP1 13 9 − (SEQ ID NO: 1043) (SEQ ID NO: 960) (SEQ ID NO: 964) miR-9GS9 miR-9 RP1 13 4 − (SEQ ID NO: 961) (SEQ ID NO: 964) miR-9GS8 miR-9 RP1 10 0 − (SEQ ID NO:962) (SEQ ID NO: 964) miR-9GS7 miR-9 RP1 16 8 − (SEQ ID NO: 963) (SEQ ID NO: 964) miR-9GS10 miR-9 RP2 13 5 − (SEQ ID NO: 960) (SEQ ID NO: 965) miR-9GS9 miR-9 RP2 14 4 − (SEQ ID NO: 961) (SEQ ID NO: 965) miR-9GS8 miR-9 RP2 10 0 − (SEQ ID NO: 962) (SEQ ID NO: 965) miR-9GS7 miR-9 RP2 17 8 − (SEQ ID NO: 963) (SEQ ID NO: 965) miR-194 miR-194GS10 miR-194RP1 9 6 − (SEQ ID NO: 1040) (SEQ ID NO: 944) (SEQ ID NO: 948) miR-194GS9 miR-194RP1 11 5 − (SEQ ID NO: 945) (SEQ ID NO: 948) miR-194GS8 miR-194RP1 13 17 + (SEQ ID NO: 946) (SEQ ID NO: 948) miR-194GS7 miR-194RP1 15 17 − (SEQ ID NO: 947) (SEQ ID NO: 948) miR-194GS10 miR-194RP2 10 6 − (SEQ ID NO: 944) (SEQ ID NO: 949) miR-194GS9 miR-194RP2 11 6 − (SEQ ID NO: 945) (SEQ ID NO: 949) miR-194GS8 miR-194RP2 13 16 − (SEQ ID NO: 946) (SEQ ID NO: 949) miR-194GS7 miR-194RP2 17 16 − (SEQ ID NO: 947) (SEQ ID NO: 949) miR-1 miR-1 GS10 miR-1 RP1 15 15 − (SEQ ID NO: 1042) (SEQ ID NO: 47) (SEQ ID NO: 959) miR-1 GS9 miR-1 RP1 17 8 − (SEQ ID NO: 956) (SEQ ID NO: 959) miR-1 GS8 miR-1 RP1 19 11 − (SEQ ID NO: 957) (SEQ ID NO: 959) miR-1 GS7 miR-1 RP1 22 11 − (SEQ ID NO: 958) (SEQ ID NO: 959) miR-1 GS10 miR-1 RP2 13 15 + (SEQ ID NO: 47) (SEQ ID NO: 48) miR-1 GS9 miR-1 RP2 15 8 − (SEQ ID NO: 956) (SEQ ID NO: 48) miR-1 GS8 miR-1 RP2 17 11 − (SEQ ID NO: 957) (SEQ ID NO: 48) miR-1 GS7 miR-1 RP2 19 10 − (SEQ ID NO: 958) (SEQ ID NO: 48) miR-206 miR-206 GS10 miR-206RP1 15 10 − (SEQ ID NO: 1041) (SEQ ID NO: 950) (SEQ ID NO: 954) miR-206 GS9 miR-206RP1 16 10 − (SEQ ID NO: 951) (SEQ ID NO: 954) miR-206 GS8 miR-206RP1 17 14 − (SEQ ID NO: 952) (SEQ ID NO: 954) miR-206 GS7 miR-206RP1 20 20 − (SEQ ID NO: 953) (SEQ ID NO: 954) miR-206 GS10 miR-206RP2 10 8 − (SEQ ID NO: 950) (SEQ ID NO: 955) miR-206 GS9 miR-206RP2 11 9 − (SEQ ID NO: 951) (SEQ ID NO: 955) miR-206 GS8 miR-206RP2 11 11 − (SEQ ID NO: 952) (SEQ ID NO: 955) miR-206 GS7 miR-206RP2 13 20 + (SEQ ID NO: 953) (SEQ ID NO: 955)

Optimal primer pairs were identified based on superior sensitivity (e.g., a preferred range between 5 and 25) and dynamic range (e.g., a preferred range between 10 and 35) characteristics. As shown above in TABLE 10, an optimal primer pair was identified for miR-194: GS8 (SEQ ID NO:946) and RP1 (SEQ ID NO:948) with a sensitivity of 13 and a dynamic range of 17. An optimal primer pair was identified for miR-1: GS10 (SEQ ID NO:47) and RP2 (SEQ ID NO:48) with a sensitivity of 13 and a dynamic range of 15. An optimal primer pair was identified for miR-206: GS7 (SEQ ID NO:953) and RP2 (SEQ ID NO:955) with a sensitivity of 13 and a dynamic range of 20. As also shown in TABLE 10, the GS primers control specificity, as shown by the significant increase in dynamic range (driven by a decrease in background) in going from GS9 to GS8 (see, e.g., miR-194).

Candidate primers designed based on the principles described above, such as the additional exemplary primers listed in TABLE 8, or other candidate primers designed using the design principles described herein, may be tested using the screening methods described above. The assays may be further optimized by using HPLC purified templates to avoid problems associated with degraded templates.

It has also been determined that microRNAs that differ from each other in sequence by only 1, 2 or 3 nucleotide changes can be readily distinguished from one another through the use of the primers designed according to the design principles and methods described herein.

While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

1. A kit for detecting at least one human microRNA selected from the group consisting of miR-21, miR-22, miR-33, miR-34a, miR-34b, miR-34c, miR-122, and miR-122a, the kit comprising at least one or more oligonucleotide primers selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:449, SEQ ID NO:450, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO: 113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:8; SEQ ID NO:20, SEQ ID NO:147 and SEQ ID NO:148.

2. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:73 and SEQ ID NO:74 for detecting miR-21.

3. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:449 and SEQ ID NO:450 for detecting miR-22.

4. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:111 and SEQ ID NO:112 for detecting miR-33.

5. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO: 113,and SEQ ID NO:114 for detecting miR-34a.

6. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:116 for detecting miR-34b.

7. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:117 and SEQ ID NO:118 for detecting miR-34c.

8. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:20 for detecting miR-122.

9. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:147 and 148 for detecting miR-122a.

10. An oligonucleotide primer for detecting a human microRNA selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:449, SEQ ID NO:450, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO: 113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:8; SEQ ID NO:20, SEQ ID NO:147 and SEQ ID NO:148.

Patent History
Publication number: 20120009580
Type: Application
Filed: Sep 22, 2011
Publication Date: Jan 12, 2012
Applicant: MERCK SHARP & DOHME CORP. (Rahway, NJ)
Inventor: Christopher K. Raymond (Seattle, WA)
Application Number: 13/240,847