Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

J Vis Exp. 2015 Apr 14;(98):52586. doi: 10.3791/52586.

Abstract

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • DNA, Complementary / chemistry
  • DNA, Complementary / genetics
  • Humans
  • MicroRNAs / blood*
  • MicroRNAs / genetics
  • MicroRNAs / isolation & purification
  • Microfluidics / methods
  • Nanotechnology / instrumentation
  • Nanotechnology / methods
  • Real-Time Polymerase Chain Reaction / instrumentation
  • Real-Time Polymerase Chain Reaction / methods*

Substances

  • DNA, Complementary
  • MicroRNAs