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Digital PCR pp 445-457 | Cite as

Quantification of Circulating MicroRNAs by Droplet Digital PCR

  • Manuela FerracinEmail author
  • Massimo NegriniEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1768)

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.

Key words

MicroRNA Serum Plasma Droplet digital PCR Diagnostics Cancer Biomarkers 

Notes

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.

References

  1. 1.
    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.CrossRefGoogle Scholar
  2. 2.
    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.CrossRefGoogle Scholar
  3. 3.
    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.PubMedCentralCrossRefGoogle Scholar
  4. 4.
    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.PubMedCentralCrossRefGoogle Scholar
  5. 5.
    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.CrossRefGoogle Scholar
  6. 6.
    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.PubMedCentralCrossRefGoogle Scholar
  7. 7.
    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.PubMedCentralCrossRefGoogle Scholar
  8. 8.
    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.PubMedCentralCrossRefGoogle Scholar
  9. 9.
    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.CrossRefGoogle Scholar
  10. 10.
    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.PubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Experimental, Diagnostic and Specialty Medicine—DIMESUniversity of BolognaBolognaItaly
  2. 2.Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental MedicineUniversity of FerraraFerraraItaly

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