B-375 Mer-idpcr: A Method for Sequence-specific Measurement of Small RNA Expression and Methylation in Liquid Biopsies

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Abstract Background Small RNAs are an important class of biomarkers in liquid biopsy for cancer diagnostics. New discoveries in the field of “epitranscriptomics” have uncovered further functional layers of tumor biology that are ignored by standard gene expression measurement methods. Therefore, there is a pressing need for sequence specific methods that can detect methylation stoichiometry on small RNAs purified from liquid biopsies in a diagnostic setting. Methods To detect small RNAs specifically, we designed structured oligonucleotide adapters that bear reverse complementary overhangs to both the 5′ and 3′ end of target RNAs. After nick-based ligation, reverse transcription under limiting dNTP conditions enables the differential processing of methylated vs unmethylated species. Finally, target specific TaqMan probes are used on a digital PCR based platform for final quantification and calculation of methylation stoichiometry. Results We show that mer-idPCR can detect target RNAs in a sequence-specific manner using synthetic cognate RNA oligos and specificity controls, such as nucleotide variants or non-target miRNAs. Detected RNA is quantified in a linear range with a limit of detection at 40 copies/µL of cDNA. Concurrently, the method can detect 2′-o-methylation in 0%–100% range with a resolution of 10%. mer-idPCR can be applied to RNA extracted from various sources including serum, plasma or whole blood stabilizing tubes. Conclusion mer-idPCR can enable the high resolution, sequence specific quantification of target small RNA species, in addition to the measurement of 2′-o-methylation site status, in small RNAs extracted from peripheral blood. We show the adapter design requirements and discuss optimizations of adapter design and reverse transcription conditions that led to improved specificity, reproducibility, and linearity of this method. mer-idPCR enables the validation and discovery of novel biomarkers for cancer detection in liquid biopsies.

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