Abstract
MicroRNAs (miRNAs) are released in the blood as cell-free molecules either linked to Ago proteins and LDL or enveloped inside exosomes and microvescicles. The amount of specific circulating microRNAs has been discovered to change accordingly to a disease state and to be potentially used as a disease biomarker. Sensitive and accurate methods for circulating microRNA quantification using probe-based or dye-based digital PCR technology have been developed. With a digital PCR system it is possible to obtain the absolute quantification of specific miRNAs, bypassing several issues related to low abundance targets and miRNA normalization. This chapter addresses the workflow and methods for miRNA assessment in biological fluids using EvaGreen-based droplet digital PCR as well as how to analyze and interpret results.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Chen X, Ba Y, Ma L, Cai X, Yin Y, Wang K et al (2008) Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 18(10):997–1006. PubMed PMID: 18766170. Epub 2008/09/04. eng.
Lawrie CH, Gal S, Dunlop HM, Pushkaran B, Liggins AP, Pulford K et al (2008) Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma. Br J Haematol 141(5):672–675. PubMed PMID: 18318758. Epub 2008/03/06. eng.
Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL et al (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 105(30):10513–10518. PubMed PMID: 18663219. Pubmed Central PMCID: 2492472. Epub 2008/07/30. eng.
Jarry J, Schadendorf D, Greenwood C, Spatz A, van Kempen LC (2014) The validity of circulating microRNAs in oncology: five years of challenges and contradictions. Mol Oncol 8(4):819–829. PubMed PMID: 24656978. Epub 2014/03/25. eng.
Witwer KW (2015) Circulating MicroRNA biomarker studies: pitfalls and potential solutions. Clin Chem 61(1):56–63. PubMed PMID: 25391989. Epub 2014/11/14. Eng.
Moldovan L, Batte KE, Trgovcich J, Wisler J, Marsh CB, Piper M (2014) Methodological challenges in utilizing miRNAs as circulating biomarkers. J Cell Mol Med 18(3):371–390. PubMed PMID: 24533657. Pubmed Central PMCID: 3943687. Epub 2014/02/19. eng.
Ferracin M, Lupini L, Salamon I, Saccenti E, Zanzi MV, Rocchi A et al (2015) Absolute quantification of cell-free microRNAs in cancer patients. Oncotarget 6(16):14545–14555. PubMed PMID: 26036630. Epub 2015/06/04. Eng.
Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ et al (2013) Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods 10(10):1003–1005. PubMed PMID: 23995387. Epub 2013/09/03. eng.
Miotto E, Saccenti E, Lupini L, Callegari E, Negrini M, Ferracin M (2014) Quantification of circulating miRNAs by droplet digital PCR: comparison of EvaGreen- and TaqMan-based chemistries. Cancer Epidemiol Biomark Prev 23(12):2638–2642. PubMed PMID: 25472671. Epub 2014/12/05. eng.
Cheng HH, Yi HS, Kim Y, Kroh EM, Chien JW, Eaton KD et al (2013) Plasma processing conditions substantially influence circulating microRNA biomarker levels. PLoS One 8(6):e64795. PubMed PMID: 23762257. Pubmed Central PMCID: 3676411. Epub 2013/06/14. eng.
Acknowledgments
The work was supported by funding from the Italian Association for Cancer Research (AIRC) to MF (MFAG 11676) and to MN (Special Program Molecular Clinical Oncology - 5 per mille n. 9980, 2010/15) and from the Italian Ministry of Instruction, University and Research FIRB 2011 (Project RBAPIIBYNP) and University of Ferrara (FAR 2012-14) to MN.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Ferracin, M., Negrini, M. (2018). Quantification of Circulating MicroRNAs by Droplet Digital PCR. In: Karlin-Neumann, G., Bizouarn, F. (eds) Digital PCR. Methods in Molecular Biology, vol 1768. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7778-9_25
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7778-9_25
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7776-5
Online ISBN: 978-1-4939-7778-9
eBook Packages: Springer Protocols