Real-time qRT-PCR assay for the detection of miRNAs using bi-directional extension sequences

Anal Biochem. 2017 Nov 1:536:32-35. doi: 10.1016/j.ab.2017.08.006. Epub 2017 Aug 12.

Abstract

Highly specific detection of miRNAs was performed using a novel bi-directional extension (BDE) assay. After reverse transcription, the cDNA was hybridized to a uniquely designed specific BDE sequence; this cDNA-BDE hybrid forms the PCR template. The PCR template was amplified in a SYBR Green-based quantitative real-time PCR. The miR-145 in human brain total RNA could be detected quantitatively in the range of seven orders of magnitude with high linearity and reproducibility. This innovative BDE 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.

Keywords: Bi-directional extension (BDE); Poly(A) tailing assay; RT-qPCR; Reverse transcription; microRNA (miRNA).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Complementary / genetics
  • Humans
  • MicroRNAs / genetics*
  • Real-Time Polymerase Chain Reaction*
  • Sensitivity and Specificity

Substances

  • DNA, Complementary
  • MIRN145 microRNA, human
  • MicroRNAs