Skip to main content
SpringerLink
Log in

Single-Cell Analysis of circRNA Using ddPCR

  • Protocol
  • First Online:
Single-Cell Assays

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2689))

Abstract

Droplet digital polymerase chain reaction (ddPCR) is a new quantitative PCR method based on water-oil emulsion droplet technology. ddPCR enables highly sensitive and accurate quantification of nucleic acid molecules, especially when their copy numbers are low. In ddPCR, a sample is fractionated into ~20,000 droplets, and every nanoliter-sized droplet undergoes PCR amplification of the target molecule. The fluorescence signals of droplets are then recorded by an automated droplet reader. Circular RNAs (circRNAs) are single-stranded, covalently closed RNA molecules that are ubiquitously expressed in animals and plants. CircRNAs are promising as biomarkers for cancer diagnosis and prognosis and as therapeutic targets or agents to inhibit oncogenic microRNAs or proteins (Kristensen LS, Jakobsen T, Hager H, Kjems J, Nat Rev Clin Oncol 19:188–206, 2022). In this chapter, the procedures for the quantitation of a circRNA in single pancreatic cancer cells using ddPCR are described.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Kristensen LS, Jakobsen T, Hager H, Kjems J (2022) The emerging roles of circRNAs in cancer and oncology. Nat Rev Clin Oncol 19(3):188–206

    Article  CAS  PubMed  Google Scholar 

  2. Baker M (2012) Digital PCR hits its stride. Nat Methods 9(6):541–544

    Article  CAS  Google Scholar 

  3. 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(3):444–449

    CAS  PubMed  Google Scholar 

  4. Vogelstein B, Kinzler Kenneth W (1999) Digital PCR. Proc Natl Acad Sci 96(16):9236–9241

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Dong L, Meng Y, Sui Z, Wang J, Wu L, Fu B (2015) Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material. Sci Rep 5:13174

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Tewhey R, Warner JB, Nakano M, Libby B, Medkova M, David PH, Kotsopoulos SK, Samuels ML, Hutchison JB, Larson JW, Topol EJ, Weiner MP, Harismendy O, Olson J, Link DR, Frazer KA (2009) Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat Biotechnol 27(11):1025–1031

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, Bright IJ, Lucero MY, Hiddessen AL, Legler TC, Kitano TK, Hodel MR, Petersen JF, Wyatt PW, Steenblock ER, Shah PH, Bousse LJ, Troup CB, Mellen JC, Wittmann DK, Erndt NG, Cauley TH, Koehler RT, So AP, Dube S, Rose KA, Montesclaros L, Wang S, Stumbo DP, Hodges SP, Romine S, Milanovich FP, White HE, Regan JF, Karlin-Neumann GA, Hindson CM, Saxonov S, Colston BW (2011) High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 83(22):8604–8610

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zonta E, Garlan F, Pécuchet N, Perez-Toralla K, Caen O, Milbury C, Didelot A, Fabre E, Blons H, Laurent-Puig P, Taly V (2016) Multiplex detection of rare mutations by picoliter droplet based digital PCR: sensitivity and specificity considerations. PLoS One 11(7):e0159094

    Article  PubMed  PubMed Central  Google Scholar 

  9. Sanders R, Mason DJ, Foy CA, Huggett JF (2013) Evaluation of digital PCR for absolute RNA quantification. PLoS One 8(9):e75296

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Men Y, Fu Y, Chen Z, Sims PA, Greenleaf WJ, Huang Y (2012) Digital polymerase chain reaction in an array of femtoliter polydimethylsiloxane microreactors. Anal Chem 84(10):4262–4266

    Article  CAS  PubMed  Google Scholar 

  11. Warren L, Bryder D, Weissman Irving L, Quake Stephen R (2006) Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. Proc Natl Acad Sci 103(47):17807–17812

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Streets AM, Huang Y (2014) Microfluidics for biological measurements with single-molecule resolution. Curr Opin Biotechnol 25:69–77

    Article  CAS  PubMed  Google Scholar 

  13. Majumdar N, Wessel T, Marks J (2015) Digital PCR modeling for maximal sensitivity, dynamic range and measurement precision. PLoS One 10(3):e0118833

    Article  PubMed  PubMed Central  Google Scholar 

  14. Whale AS, Huggett JF, Tzonev S (2016) Fundamentals of multiplexing with digital PCR. Biomol Detect Quantif 10:15–23

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Whale AS, Cowen S, Foy CA, Huggett JF (2013) Methods for applying accurate digital PCR analysis on low copy DNA samples. PLoS One 8(3):e58177

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ludlow AT, Robin JD, Sayed M, Litterst CM, Shelton DN, Shay JW, Wright WE (2014) Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution. Nucleic Acids Res 42(13):e104–e104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Tian H, Sun Y, Liu C, Duan X, Tang W, Li Z (2016) Precise quantitation of MicroRNA in a single cell with droplet digital PCR based on ligation reaction. Anal Chem 88(23):11384–11389

    Article  CAS  PubMed  Google Scholar 

  18. O’Hara R, Tedone E, Ludlow A, Huang E, Arosio B, Mari D, Shay JW (2019) Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution. Genome Res 29(11):1878–1888

    Article  PubMed  PubMed Central  Google Scholar 

  19. Zhu D, Zhao Z, Cui G, Chang S, Hu L, See YX, Lim MGL, Guo D, Chen X, Poudel B, Robson P, Luo Y, Cheung E (2018) Single-cell transcriptome analysis reveals estrogen signaling coordinately augments one-carbon, polyamine, and purine synthesis in breast cancer. Cell Rep 25(8):2285–2298.e2284

    Article  CAS  PubMed  Google Scholar 

  20. Kojabad AA, Farzanehpour M, Galeh HEG, Dorostkar R, Jafarpour A, Bolandian M, Nodooshan MM (2021) Droplet digital PCR of viral DNA/RNA, current progress, challenges, and future perspectives. J Med Virol 93(7):4182–4197

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Liu X, Feng J, Zhang Q, Guo D, Zhang L, Suo T, Hu W, Guo M, Wang X, Huang Z, Xiong Y, Chen G, Chen Y, Lan K (2020) Analytical comparisons of SARS-COV-2 detection by qRT-PCR and ddPCR with multiple primer/probe sets. Emerg Microbes Infect 9(1):1175–1179

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Watanabe M, Kawaguchi T, Isa S, Ando M, Tamiya A, Kubo A, Saka H, Takeo S, Adachi H, Tagawa T, Kakegawa S, Yamashita M, Kataoka K, Ichinose Y, Takeuchi Y, Sakamoto K, Matsumura A, Koh Y (2015) Ultra-sensitive detection of the pretreatment EGFR T790M mutation in non-small cell lung cancer patients with an EGFR-activating mutation using droplet digital PCR. Clin Cancer Res 21(15):3552–3560

    Article  CAS  PubMed  Google Scholar 

  23. Olmedillas-López S, García-Arranz M, García-Olmo D (2017) Current and emerging applications of droplet digital PCR in oncology. Mol Diagn Ther 21(5):493–510

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Oral and Systemic Health Sciences, School of Dentistry, University of California, Los Angeles, CA, USA

    Jiayi Peng, Feng Li, Xiangdong Xu & Shen Hu

  2. Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA, USA

    Shen Hu

  3. California NanoSystems Institute, University of California, Los Angeles, CA, USA

    Shen Hu

Authors
  1. Jiayi Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Feng Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiangdong Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shen Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shen Hu .

Editor information

Editors and Affiliations

  1. Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada

    Paul C.H. Li

  2. Division of Life Science, Hong Kong University of Science and Technology, Kowloon, Hong Kong, China

    Angela Ruohao Wu

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Peng, J., Li, F., Xu, X., Hu, S. (2023). Single-Cell Analysis of circRNA Using ddPCR. In: Li, P.C., Wu, A.R. (eds) Single-Cell Assays. Methods in Molecular Biology, vol 2689. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3323-6_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3323-6_13

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3322-9

  • Online ISBN: 978-1-0716-3323-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation