A simplified protocol for profiling heparin-contaminated circulating miRNAs: by microfluidic array
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A simplified protocol for profiling heparin‑contaminated circulating miRNAs: by microfluidic array Jesse D. Armitage1,2 · Erin M. Bolitho3 · Yuben P. Moodley1,2,4 · Dino B. A. Tan1,2 Received: 12 May 2020 / Accepted: 29 October 2020 © Springer Nature B.V. 2020
Abstract Peripheral blood is a valuable, non-invasive source of biomarkers which include circulating miRNAs. Using microfluidic array-based techniques, miRNAs can be successfully measured in small amounts of blood plasma (< 0.5 mL) using cDNA pre-amplification. However, the use of heparin-based anticoagulants for blood collection hinders the detection of circulating miRNAs due to its inhibitory effect on PCR components. Although pre-treatment with heparinase have been shown to overcome heparin contamination in blood, its effect has not been described in array-based analyses or more sensitive applications with smaller sample volumes (i.e. 200 μL plasma) requiring pre-amplification. We show that the treatment of miRNA extracted from heparinised plasma with an optimised concentration of Bacteroides heparinase I prior to cDNA preamplification dramatically improves the number of detectable miRNA from 2 to 67 targets on the TaqMan® Array Human MicroRNA Cards. Furthermore, the titrated amount of heparinase (3 U) gave the best miRNA detection compared to those used in previous studies (6–24 U). This study provides novel data which demonstrates that heparinase treatment is compatible with protocols that involve pre-amplification of cDNA and microfluidic array-based techniques. This an improved methodology that permits miRNA-based biomarker analysis from small volume of heparinised plasma. Keywords miRNA · Taqman · RT-qPCR · Heparin · Biomarker · Microfluidic array
Introduction MicroRNAs (miRNAs) are small, non-coding RNAs that mediate the degradation of mRNAs by binding to their 3’ untranslated region [1]. The prognostic and diagnostic value of blood-based miRNAs has gained considerable momentum in recent years, where microfluidic arrays are useful in canvassing hundreds of miRNAs for their biomarker potential [2]. High throughput screening of circulating miRNAs remains a challenge when working with limited volumes * Dino B. A. Tan [email protected] 1
Centre 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
2
Stem Cell Unit, Institute for Respiratory Health, Perth, WA, Australia
3
Research Centre, Royal Perth Hospital, Perth, WA, Australia
4
Department of Respiratory Medicine, Fiona Stanley Hospital, Murdoch, WA, Australia
of blood plasma (< 0.5 mL), however an initial cDNA preamplification step can be incorporated to improve the starting concentration of template cDNA prior to the quantitative polymerase chain reaction (qPCR). Collection of peripheral blood into heparin-coated tubes is a common laboratory practice; however downstream RNA/DNA analysis using heparinised plasma is ge
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