Abstract
Cell-to-cell communication involves the release of biological molecules into the extracellular space and their uptake by recipient cells. These molecules include RNA that can modulate cellular signaling and biological processes. To study extracellular RNA, highly sensitive and precise methods for their detection are needed. Digital polymerase chain reaction (dPCR) can be a useful method for detecting and analyzing extracellular RNA. The sensitivity of digital PCR can exceed that of quantitative PCR for low abundance targets such as extracellular RNA.
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References
Morley AA (2014) Digital PCR: a brief history. Biomol Detect Quantif 1(1):1–2. https://doi.org/10.1016/j.bdq.2014.06.001
Bellingham SA, Shambrook M, Hill AF (2017) Quantitative analysis of exosomal miRNA via qPCR and digital PCR. Methods Mol Biol 1545:55–70. https://doi.org/10.1007/978-1-4939-6728-5_5
Yan IK, Wang X, Asmann YW, Haga H, Patel T (2016) Circulating extracellular RNA markers of liver regeneration. PLoS One 11(7):e0155888. https://doi.org/10.1371/journal.pone.0155888
Takahashi K, Yan IK, Kim C, Kim J, Patel T (2014) Analysis of extracellular RNA by digital PCR. Front Oncol 4:129. https://doi.org/10.3389/fonc.2014.00129
Taylor SC, Laperriere G, Germain H (2017) Droplet digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Sci Rep 7(1):2409. https://doi.org/10.1038/s41598-017-02217-x
McDermott GP, Do D, Litterst CM, Maar D, Hindson CM, Steenblock ER, Legler TC, Jouvenot Y, Marrs SH, Bemis A, Shah P, Wong J, Wang S, Sally D, Javier L, Dinio T, Han C, Brackbill TP, Hodges SP, Ling Y, Klitgord N, Carman GJ, Berman JR, Koehler RT, Hiddessen AL, Walse P, Bousse L, Tzonev S, Hefner E, Hindson BJ, Cauly TH 3rd, Hamby K, Patel VP, Regan JF, Wyatt PW, Karlin-Neumann GA, Stumbo DP, Lowe AJ (2013) Multiplexed target detection using DNA-binding dye chemistry in droplet digital PCR. Anal Chem 85(23):11619–11627. https://doi.org/10.1021/ac403061n
Huggett JF, Foy CA, Benes V, Emslie K, Garson JA, Haynes R, Hellemans J, Kubista M, Mueller RD, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT, Bustin SA (2013) The digital MIQE guidelines: minimum information for publication of quantitative digital PCR experiments. Clin Chem 59(6):892–902. https://doi.org/10.1373/clinchem.2013.206375
Huggett JF, Cowen S, Foy CA (2015) Considerations for digital PCR as an accurate molecular diagnostic tool. Clin Chem 61(1):79–88. https://doi.org/10.1373/clinchem.2014.221366
Acknowledgments
This work was supported by the National Institutes of Health (USA) Office of the Director through grant UH3 TR000884. We acknowledge the expert assistance of Caitlyn Foerst and thank the members of our laboratories for their contributions.
Disclosures: None.
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Yan, I.K., Lohray, R., Patel, T. (2018). Droplet Digital PCR for Quantitation of Extracellular RNA. In: Patel, T. (eds) Extracellular RNA. Methods in Molecular Biology, vol 1740. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7652-2_12
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DOI: https://doi.org/10.1007/978-1-4939-7652-2_12
Publisher Name: Humana Press, New York, NY
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