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

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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 pre-amplification 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.

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Acknowledgements

The authors thank all donors used in this study and nursing staff at Royal Perth Hospital for collection of blood samples. The authors also thank A/Prof. Richard Allcock and Dr. Belinda Guo from the LotteryWest State Biomedical Facility Genomics (Perth, Australia) for technical assistance on the Viia7™ PCR system.

Funding

This study was supported by funding from a Medical Research Foundation grant (Royal Perth Hospital, Australia).

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Authors

Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by JA, EB and DT. The first draft of the manuscript was written by JA and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Dino B. A. Tan.

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Conflicts of interest

The authors in this study have no conflicts of interest.

Ethics approval

The study was approved by the Royal Perth Hospital ethics committee (approval no. EC2012/103)

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All patients had provided written informed consent.

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All patients had provided written informed consent.

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Armitage, J.D., Bolitho, E.M., Moodley, Y.P. et al. A simplified protocol for profiling heparin-contaminated circulating miRNAs: by microfluidic array. Mol Biol Rep 47, 9973–9977 (2020). https://doi.org/10.1007/s11033-020-05964-9

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  • DOI: https://doi.org/10.1007/s11033-020-05964-9

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