Efficient and accurate analysis of microRNA using a specific extension sequence

Efficient and accurate analysis of microRNA using a specific extension sequence We present here on an innovative assay for detecting miRNAs using a uniquely designed specific extension sequence that provides high efficiency and accuracy. This assay consists of poly(A) tailing and reverse transcription followed by real-time PCR. In the first step of this reaction, target miRNAs are poly(A) tailed by poly(A) polymerase followed by cDNA synthesis using poly(T) adaptors. In the second step, cDNA is hybridized to the 3′-end of a specific extension sequence that contains part of a miRNA sequence; this cDNA-specific extension sequence hybrid forms the novel PCR template. The PCR template is amplified in a SYBR Green-based quantitative real-time PCR with universal forward and reverse primers. The miR-106b in human brain total RNA could be detected quantitatively in the range of seven orders of magnitude with high linearity and reproducibility. This innovative extension-based assay has several performance advantages over the poly(A) tailing method that include lower CT values, clear gel electrophoresis images, and distinct nucleotide peaks in sequencing chromatograms. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Biology Reports Springer Journals

Efficient and accurate analysis of microRNA using a specific extension sequence

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Publisher
Springer Netherlands
Copyright
Copyright © 2018 by Springer Science+Business Media B.V., part of Springer Nature
Subject
Life Sciences; Animal Biochemistry; Animal Anatomy / Morphology / Histology
ISSN
0301-4851
eISSN
1573-4978
D.O.I.
10.1007/s11033-018-4200-1
Publisher site
See Article on Publisher Site

Abstract

We present here on an innovative assay for detecting miRNAs using a uniquely designed specific extension sequence that provides high efficiency and accuracy. This assay consists of poly(A) tailing and reverse transcription followed by real-time PCR. In the first step of this reaction, target miRNAs are poly(A) tailed by poly(A) polymerase followed by cDNA synthesis using poly(T) adaptors. In the second step, cDNA is hybridized to the 3′-end of a specific extension sequence that contains part of a miRNA sequence; this cDNA-specific extension sequence hybrid forms the novel PCR template. The PCR template is amplified in a SYBR Green-based quantitative real-time PCR with universal forward and reverse primers. The miR-106b in human brain total RNA could be detected quantitatively in the range of seven orders of magnitude with high linearity and reproducibility. This innovative extension-based assay has several performance advantages over the poly(A) tailing method that include lower CT values, clear gel electrophoresis images, and distinct nucleotide peaks in sequencing chromatograms.

Journal

Molecular Biology ReportsSpringer Journals

Published: May 30, 2018

References

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