Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification

0482563 - BTÚ 2018 RIV GB eng J - Journal Article
Androvič, Peter - Valihrach, Lukáš - Elling, J. - Sjöback, R. - Kubista, Mikael
Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification.
Nucleic Acids Research. Roč. 45, č. 15 (2017), č. článku e144. ISSN 0305-1048. E-ISSN 1362-4962
R&D Projects: GA ČR(CZ) GA16-10214S; GA ČR(CZ) GA16-10214S; GA MŠMT(CZ) ED1.1.00/02.0109
Institutional support: RVO:86652036
Keywords : rolling circle amplification * microrna biogenesis pathways * real-time pcr * neurodegenerative diseases
OECD category: Genetics and heredity (medical genetics to be 3)
Impact factor: 11.561, year: 2017

MicroRNAs are a class of small non-coding RNAs that serve as important regulators of gene expression at the posttranscriptional level. They are stable in body fluids and pose great potential to serve as biomarkers. Here, we present a highly specific, sensitive 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 mismatched nucleotide. Two-tailed RT-qPCR has a dynamic range of seven logs and a sensitivity sufficient to detect down to ten target miRNA molecules. It is capable to capture the full isomiR repertoire, leading to accurate representation of the complete 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 (r(2) = 0.985). Moreover, Two-tailed RT-qPCR allows for rapid testing with a total analysis time of less than 2.5 hours.
Permanent Link: http://hdl.handle.net/11104/0277987