Abstract: Provided herein is a method for detecting at least two distinct oligonucleotides of equal length in parallel from one biological sample, comprising the steps of providing a biological sample containing or suspected of containing oligonucleotides of interest; forming a hybridization mixture using at least two fluorescently labelled detection molecules with different surface charges; separating the detection molecules hybridized to the oligonucleotides by anion exchange HPLC; and detecting the hybridized detection molecule-oligonucleotide moieties by quantitative fluorescence readout. In a further aspect, a kit comprising at least two detection molecules is provided. In another aspect, provided herein is the use of at least two detection molecules with different surface charges for quantitatively detecting at least two distinct oligonucleotides of equal length in parallel from one biological sample.

Abstract: In a first aspect, the invention relates to a method for detecting at least two distinct oligonucleotides of equal length in parallel from one biological sample, said method comprising the steps of providing a biological sample containing or suspected of containing said oligonucleotides of interest; forming a hybridization mixture using at least two fluorescently labelled detection molecules with different surface charges; separating the detection molecules hybridised to said oligonucleotides by anion exchange HPLC; and detecting the hybridized detection molecule-oligonucleotide moieties by means of quantitative fluorescence readout. In a further aspect, the invention relates to a kit comprising at least two detection molecules as defined in the present invention. In another aspect, the invention relates to the use of at least two detection molecules with different surface charges for quantitatively detecting at least two distinct oligonucleotides of equal length in parallel from one biological sample.