Lukas Valihrach 1, Peter Androvic1, Julie Elling2, Robert Sjoback2, Mikael Kubista1,2
1 Institute of Biotechnology AS CR, Czech Republic
2 TATAA Biocenter AB, Sweden
MicroRNAs are a class of small non-coding RNAs that serve as important regulators of gene expression at the posttranscriptional level. MiRNAs are stable in body fluids and pose great potential to serve as biomarkers. Here, we present a highly specific, sensi- tive and cost-effective system to quantify miRNA expression based on two-step RT-qPCR with SYBR-green detection chemistry called Two-tailed RT-qPCR. It takes advantage of novel, target-specific primers for reverse transcription composed of two hemiprobes complementary to two different parts of the targeted miRNA, connected by a hairpin structure. The introduction of a second probe ensures high sensitivity and enables discrimination of highly homologous miRNAs irrespectively of the position of the mis- matched nucleotide. Two-tailed RT-qPCR has a dynamic range of 8 logs and a sensitivity sufficient to detect down to a hun- dred of target miRNA molecules. It is capable to capture the full isomiR repertoire, leading to accurate representation of the com- plete miRNA content in a sample. The reverse transcription step can be multiplexed and the miRNA profiles measured with Two-tailed RT-qPCR show excellent correlation with the industry standard TaqMan miRNA assays (R2 = 0.985). Moreover, Two-tailed RT-qPCR allows for rapid testing with a total analysis time of less than 2.5 hours.
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