Detection of microRNAs Using Chip-Based QuantStudio 3D Digital PCR

  • Cristina Borzi
  • Linda Calzolari
  • Davide Conte
  • Gabriella SozziEmail author
  • Orazio Fortunato
Part of the Methods in Molecular Biology book series (MIMB, volume 1580)


Digital PCR (dPCR) is an innovative approach for detection and quantification of nucleic acid that offers an alternative method to conventional real-time quantitative PCR for absolute quantification. dPCR is a highly precise and sensitive technique that does not require a standard reference, making it a suitable method for the detection of microRNAs. The potential of these small noncoding RNA as biomarkers is on the rise, especially due to their presence in body fluids, making them easily accessible. Nevertheless, the problem of lack of consensus regarding an optimal method for miRNAs normalization compromises their use. Here, we describe an innovative method for the absolute quantification of miRNAs across different types of biological samples using a chip-based platform, the QuantStudio 3D digital PCR.

Key words

Digital PCR Absolute quantification miRNAs 



This work was supported by AIRC (Associazione Italiana per la Ricerca sul Cancro): IG14318 to G.S., and 12162 (Special Program “Innovative Tools for Cancer Risk Assessment and early Diagnosis,” 5 × 1000); Italian Ministry of Health RF-2010-2306232 to G.S.


  1. 1.
    Sykes PJ, Neoh SH, Brisco MJ, Hughes E, Condon J, Morley AA (1992) Quantitation of targets for PCR by use of limiting dilution. Biotechniques 13:444–449PubMedGoogle Scholar
  2. 2.
    Warren L, Bryder D, Weissman IL, Quake SR (2006) Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. Proc Natl Acad Sci U S A 103:17807–17812CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Devonshire AS, Sanders R, Wilkes TM, Taylor MS, Foy CA, Huggett JF (2013) Application of next generation qPCR and sequencing platforms to mRNA biomarker analysis. Methods 59:89–100CrossRefPubMedGoogle Scholar
  4. 4.
    Kreutz JE, Munson T, Huynh T, Shen F, Du W, Ismagilov RF (2011) Theoretical design and analysis of multivolume digital assays with wide dynamic range validated experimentally with microfluidic digital PCR. Anal Chem 83:8158–8168CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Sundberg SO, Wittwer CT, Gao C, Gale BK (2010) Spinning disk platform for microfluidic digital polymerase chain reaction. Anal Chem 82:1546–1550CrossRefPubMedGoogle Scholar
  6. 6.
    Conte D, Verri C, Borzi C, Suatoni P, Pastorino U, Sozzi S, Fortunato O (2015) Novel method to detect microRNAs using chip-based QuantStudio 3D digital PCR. BMC Genomics 16:849CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Hatch AC, Fisher JS, Tovar AR, Hsieh AT, Lin R, Pentoney SL et al (2011) 1-million droplet array with wide-field fluorescence imaging for digital PCR. Lab Chip 11:3838–3845CrossRefPubMedGoogle Scholar
  8. 8.
    Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ et al (2011) Highthroughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 83:8604–8610CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Li M, Diehl F, Dressman D, Vogelstein B, Kinzler KW (2006) BEAMing up for detection and quantification of rare sequence variants. Nat Methods 3:95–97CrossRefPubMedGoogle Scholar
  10. 10.
    Zhong Q, Bhattacharya S, Kotsopoulos S, Olson J, Taly V, Griffiths AD et al (2011) Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR. Lab Chip 11:2167–2174CrossRefPubMedGoogle Scholar
  11. 11.
    Sanders R, Huggett JF, Bushell CA, Cowen S, Scott DJ, Foy CA (2011) Evaluation of digital PCR for absolute DNA quantification. Anal Chem 83:6474–6484CrossRefPubMedGoogle Scholar
  12. 12.
    Dodd DW, Gagnon KT, Corey DR (2013) Digital quantitation of potential therapeutic target RNAs. Nucleic Acid Ther 23:188–194CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Shen J, Jiang F (2012) Applications of MicroRNAs in the Diagnosis and Prognosis of Lung Cancer. Expert Opin Med Diagn 6:197–207CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Shen J, Stass SA, Jiang F (2013) MicroRNAs as potential biomarkers in human solid tumors. Cancer Lett 329:125–136CrossRefPubMedGoogle Scholar
  15. 15.
    Gutierrez-Aguirre I, Racki N, Dreo T, Ravnikar M (2015) Droplet digital PCR for absolute quantification of pathogens. Methods Mol Biol 1302:331–347CrossRefPubMedGoogle Scholar
  16. 16.
    Pekin D, Skhiri Y, Baret JC, Le Corre D, Mazutis L, Salem CB et al (2011) Quantitative and sensitive detection of rare mutations using droplet-based microfluidics. Lab Chip 11:2156–2166CrossRefPubMedGoogle Scholar
  17. 17.
    Marques FZ, Prestes PR, Pinheiro LB, Scurrah K, Emslie KR, Tomaszewski M et al (2014) Measurement of absolute copy number variation reveals association with essential hypertension. BMC Med Genomics 7:44CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Ludlow AT, Robin JD, Sayed M, Litterst CM, Shelton DN, Shay JW et al (2014) Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution. Nucleic Acids Res 42:e104CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Ma J, Li N, Guarnera M, Jiang F (2013) Quantification of plasma miRNAs by digital PCR for cancer diagnosis. Biomark Insights 8:127–136PubMedPubMedCentralGoogle Scholar
  20. 20.
    Li N, Ma J, Guarnera MA, Fang H, Cai L, Jiang F (2014) Digital PCR quantification of miRNAs in sputum for diagnosis of lung cancer. J Cancer Res Clin Oncol 140:145–150CrossRefPubMedGoogle Scholar
  21. 21.
    Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297CrossRefPubMedGoogle Scholar
  22. 22.
    Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ (2004) Processing of primary microRNAs by the microprocessor complex. Nature 432:231–235CrossRefPubMedGoogle Scholar
  23. 23.
    Croce CM (2009) Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet 10:704–714CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Shen J, Todd NW, Zhang H, Yu L, Lingxiao X, Mei Y et al (2011) Plasma microRNAs as potential biomarkers for non-small-cell lung cancer. Lab Invest 91:579–587CrossRefPubMedGoogle Scholar
  25. 25.
    Hu Z, Chen X, Zhao Y, Tian T, Jin G, Shu Y et al (2010) Serum microRNA signatures identified in a genome-wide serum microRNA expression profiling predict survival of non-small-cell lung cancer. J Clin Oncol 28:1721–1726CrossRefPubMedGoogle Scholar
  26. 26.
    Hanke M, Hoefig K, Merz H, Feller AC, Kausch I, Jocham D et al (2010) A robust methodology to study urine microRNA as tumor marker: microRNA-126 and microRNA-182 are related to urinary bladder cancer. Urol Oncol 28:655–661CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Cristina Borzi
    • 1
  • Linda Calzolari
    • 1
  • Davide Conte
    • 1
  • Gabriella Sozzi
    • 1
    Email author
  • Orazio Fortunato
    • 1
  1. 1.Tumor Genomics Unit, Department of Experimental Oncology and Molecular MedicineFondazione IRCCS Istituto Nazionale dei TumoriMilanItaly

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