Mikael Kubista1,2, Robert Sjöback1, Andrei Herdean1, Peter Androvic2, Lukas Valihrach2
1) TATAA Biocenter AB, Sweden;
2) Institute of Biotechnology, Czech Academy of Sciences;
We present a highly specific, sensitive and cost-effective system to quantify miRNA expression based on novel chemistry called Two-tailed PCR. Two-tailed PCR takes advantage of 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 mismatched nucleotide. Two-tailed RT-qPCR has a dynamic range of 7 logs and a sensitivity sufficient to detect less than ten target miRNA molecules. The reverse transcription step can be multiplexed and it allows for rapid testing with a total analysis time of less than 2.5 hours. Several applications will be presented including a quality control panel for liquid biopsy samples and 1-tube combined analysis of mRNA and microRNA.
P. Androvic, L. Valihrach, J. Elling, R. Sjöback, M. Kubista. Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification. Nucleic Acids Research, Volume 45, Issue 15, 6 September 2017, Pages e144
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