Vol.:(0123456789) 1 3 Molecular Biology Reports https://doi.org/10.1007/s11033-020-05964-9 SHORT COMMUNICATION A simplifed protocol for profling heparin‑contaminated circulating miRNAs: by microfuidic array Jesse D. Armitage 1,2  · Erin M. Bolitho 3  · Yuben P. Moodley 1,2,4  · Dino B. A. Tan 1,2 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 microfuidic array-based techniques, miRNAs can be successfully measured in small amounts of blood plasma (< 0.5 mL) using cDNA pre-amplifcation. However, the use of heparin-based anticoagulants for blood collection hinders the detection of circulat- ing miRNAs due to its inhibitory efect on PCR components. Although pre-treatment with heparinase have been shown to overcome heparin contamination in blood, its efect has not been described in array-based analyses or more sensitive applications with smaller sample volumes (i.e. 200 μL plasma) requiring pre-amplifcation. We show that the treatment of miRNA extracted from heparinised plasma with an optimised concentration of Bacteroides heparinase I prior to cDNA pre- amplifcation 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-amplifcation of cDNA and microfuidic array-based techniques. This an improved methodol- ogy that permits miRNA-based biomarker analysis from small volume of heparinised plasma. Keywords miRNA · Taqman · RT-qPCR · Heparin · Biomarker · Microfuidic 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 momen- tum in recent years, where microfuidic arrays are useful in canvassing hundreds of miRNAs for their biomarker poten- tial [2]. High throughput screening of circulating miRNAs remains a challenge when working with limited volumes of blood plasma (< 0.5 mL), however an initial cDNA pre- amplifcation step can be incorporated to improve the start- ing 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 generally not recommended given that heparin binds and inhibits the activity of qPCR enzymes such as DNA polymerase and reverse transcriptase [3, 4]. Although previous studies have shown that heparin contamination can be overcome by treatment of circulat- ing nucleic acids with heparinase [5–8], its efect has not been described in array-based analyses or more sensitive applications with smaller sample volumes requiring pre- amplifcation. Hence, we examined the efect of hepari- nase treatment on the detection of miRNAs from 200 μL of heparinised blood plasma using a protocol that includes cDNA pre-amplifcation prior to qPCR by TaqMan ® Array Human MicroRNA Cards. This study outlines an important methodology that will enable investigators to overcome the detrimental efects of heparinised blood on downstream PCR-based array analyses. * Dino B. A. Tan dino.tan@uwa.edu.au 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