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Updated: Dec 1, 2025

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

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

Published on: April 14, 2015

A simplified protocol for profiling heparin-contaminated circulating miRNAs: by microfluidic array.

Jesse D Armitage^1,2, Erin M Bolitho³, Yuben P Moodley^1,2,4

¹Centre for Respiratory Health, School of Biomedical Sciences, University of Western Australia, Level 5, Harry Perkins Institute of Medical Research, 6 Verdun Street, Nedlands, Perth, WA, 6000, Australia.

Molecular Biology Reports

|November 10, 2020

Summary

This summary is machine-generated.

Heparin in blood collection inhibits microRNA detection. Treating plasma with Bacteroides heparinase I before cDNA pre-amplification significantly boosts detectable microRNA targets using array-based methods.

Keywords:

Biomarker Heparin Microfluidic array RT-qPCR Taqman miRNA

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Last Updated: Dec 1, 2025

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

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Published on: April 14, 2015

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

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Published on: August 3, 2011

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

Published on: January 22, 2014

Area of Science:

Biochemistry
Molecular Biology
Biomarker Discovery

Background:

Peripheral blood is a non-invasive source for circulating microRNA (miRNA) biomarkers.
Microfluidic array-based techniques allow miRNA detection in small plasma volumes (< 0.5 mL) with cDNA pre-amplification.
Heparin anticoagulants inhibit PCR, hindering miRNA detection in collected blood samples.

Purpose of the Study:

To evaluate the efficacy of heparinase treatment on miRNA detection in small volumes of heparinized plasma using array-based analysis with pre-amplification.
To optimize heparinase concentration for improved miRNA target identification.

Main Methods:

Extraction of miRNA from heparinized plasma.
Treatment with optimized concentrations of Bacteroides heparinase I.
cDNA pre-amplification followed by TaqMan® Array Human MicroRNA Cards analysis.

Main Results:

Heparinase treatment dramatically increased detectable miRNA targets from 2 to 67.
An optimized concentration of 3 U of heparinase yielded the best miRNA detection.
Heparinase treatment is compatible with pre-amplification and microfluidic array-based techniques.

Conclusions:

Heparinase treatment is a viable method to overcome heparin inhibition in plasma.
This optimized methodology enables robust miRNA biomarker analysis from small volumes of heparinized plasma.
The findings support the use of heparinized blood for sensitive miRNA profiling.