Abstract: The present invention relates to a method for amplifying at least one target RNA in a fixed and, optionally, expanded biological sample. In an embodiment of the invention, the method comprises incubating the fixed biological sample with a pair of polynucleotides complementary to non-overlapping and proximal sequences of a target RNA, wherein the polynucleotide pair hybridizes to the target RNA; ligating the polynucleotide pair using a ligase; and amplifying the ligation product. The invention further provides methods for detecting and optionally quantifying and/or sequencing the amplification product. As the method comprises hybridizing polynucleotide pairs to a target RNA in a fixed biological sample, the target RNA can be hybridized in situ.

Abstract: The present invention provides biological samples of interest that have been iteratively expanded in a method referred to herein as iterative direct expansion microscopy (id-ExM). In the id-ExM method, biological samples of interest are permeated with a swellable material that results in the sample becoming embedded in the swellable material, and then the sample can be expanded isotropically in three dimensions. The process of iteratively expanding the samples can be applied to expand samples one or more additional times such that, for example, a 5-fold expanded sample can be expanded again to achieve high expansion factors, for example, 20× to 100× or more linear expansion.

Abstract: The present invention relates to a method for amplifying at least one target RNA in a fixed and, optionally, expanded biological sample. In an embodiment of the invention, the method comprises incubating the fixed biological sample with a pair of polynucleotides complementary to non-overlapping and proximal sequences of a target RNA, wherein the polynucleotide pair hybridizes to the target RNA; ligating the polynucleotide pair using a ligase; and amplifying the ligation product. The invention further provides methods for detecting and optionally quantifying and/or sequencing the amplification product. As the method comprises hybridizing polynucleotide pairs to a target RNA in a fixed biological sample, the target RNA can be hybridized in situ.