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

Published on: April 14, 2015

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Quantification of microRNA in plasma using probe based TaqMan assays: is microRNA purification required?

Helle Glud Binderup1,2, Jonna Skov Madsen3,4, Claus Lohman Brasen3,4

  • 1Biochemistry and Immunology, Lillebaelt Hospital, Sygehusvej 1, 6000, Kolding, Denmark. helle.glud.binderup@rsyd.dk.

BMC Research Notes
|May 12, 2019
PubMed
Abstract

Insights

Directly quantifying circulating microRNAs using TaqMan assays in platelet-poor plasma was unsuccessful. This finding highlights challenges in optimizing microRNA biomarker analysis without a purification step.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biomarker Discovery

Background:

  • Circulating microRNAs show potential as disease biomarkers.
  • Reproducibility issues in microRNA analysis are partly due to preanalytical factors.
  • MicroRNA purification is a significant source of assay variation.

Purpose of the Study:

  • To evaluate the feasibility of direct circulating microRNA quantification without purification.
  • To adapt direct reverse transcription methods for use with TaqMan assays in platelet-poor plasma.

Main Methods:

  • Investigated direct quantification of microRNAs (miR-92a, miR-16, miR-126).
  • Utilized platelet-poor plasma and TaqMan assays.
  • Attempted adaptation of direct reverse transcription protocols.

Main Results:

  • Direct quantification of selected microRNAs in platelet-poor plasma using TaqMan assays yielded invalid results.
  • The adapted procedures were not successful for direct microRNA analysis.

Conclusions:

  • Direct quantification of circulating microRNAs in platelet-poor plasma with TaqMan assays is not currently feasible.
  • Further optimization is needed to overcome challenges in direct microRNA biomarker analysis.
Keywords:
Direct plasma RT-qPCRMicroRNASample preparationTaqMan assays

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