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

Abstract:

: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 (r2 = 0.985). Moreover, Two-tailed RT-qPCR allows for rapid testing with a total analysis time of less than 2.5 hours.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Androvic P,Valihrach L,Elling J,Sjoback R,Kubista M

doi

10.1093/nar/gkx588

subject

Has Abstract

pub_date

2017-09-06 00:00:00

pages

e144

issue

15

eissn

0305-1048

issn

1362-4962

pii

3958703

journal_volume

45

pub_type

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