Abstract
Transcript analysis is a routinely used method to assess the expression profile of progenitor cells at different stages starting from their isolation to differentiation into specific lineages. It is a powerful way to understand similarities and differences between different cell types as well to estimate successful differentiation process. Transcript measurement is most commonly done using polymerase chain reaction (PCR) but other methods such as in situ hybridization, RNA sequencing are available. The quantitative PCR using TaqMan chemistry is a highly sensitive and reproducible method that measures gene transcripts as a relative abundance. With recent advances in technology, absolute quantitation of genes to single copy level is possible using digital PCR platforms.
Digital PCR is an improved method of PCR in which a single reaction is partitioned into multiple mini reactions. Gene transcripts are measured in each of these mini reactions thereby improving assay sensitivity and making absolute quantitation possible. Here we describe the generation of human islet-derived progenitor cells and measuring gene transcripts in these cells at different passages using digital droplet PCR.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Sykes PJ et al (1992) Quantitation of targets for PCR by use of limiting dilution. BioTechniques 13(3):444–449
Saiki RK et al (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239(4839):487–491
Morley AA (2014) Digital PCR: a brief history. Biomol Detect Quantif 1(1):1–2
Vogelstein B, Kinzler KW (1999) Digital PCR. Proc Natl Acad Sci U S A 96(16):9236–9241
Manoj P (2016) Droplet digital PCR technology promises new applications and research areas. Mitochondrial DNA A DNA Mapp Seq Anal 27(1):742–746
Hindson BJ et al (2011) High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 83(22):8604–8610
Pohl G, Shih Ie M (2004) Principle and applications of digital PCR. Expert Rev Mol Diagn 4(1):41–47
Huggett JF, Whale A (2013) Digital PCR as a novel technology and its potential implications for molecular diagnostics. Clin Chem 59(12):1691–1693
Whale AS et al (2013) Methods for applying accurate digital PCR analysis on low copy DNA samples. PLoS One 8(3):e58177
Sanders R et al (2011) Evaluation of digital PCR for absolute DNA quantification. Anal Chem 83(17):6474–6484
Sanders R et al (2013) Evaluation of digital PCR for absolute RNA quantification. PLoS One 8(9):e75296
Dingle TC et al (2013) Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances. Clin Chem 59(11):1670–1672
Gershengorn MC et al (2005) Are better islet cell precursors generated by epithelial-to-mesenchymal transition? Cell Cycle 4(3):380–382
Davani B et al (2009) Human islet-derived precursor cells can cycle between epithelial clusters and mesenchymal phenotypes. J Cell Mol Med 13(8B):2570–2581
Joglekar MV, Hardikar AA (2010) Epithelial-to-mesenchymal transition in pancreatic islet beta cells. Cell Cycle 9(20):4077–4079
Bar-Nur O et al (2011) Epigenetic memory and preferential lineage-specific differentiation in induced pluripotent stem cells derived from human pancreatic islet beta cells. Cell Stem Cell 9(1):17–23
Gershengorn MC et al (2004) Epithelial-to-mesenchymal transition generates proliferative human islet precursor cells. Science 306(5705):2261–2264
Russ HA et al (2008) In vitro proliferation of cells derived from adult human beta-cells revealed by cell-lineage tracing. Diabetes 57(6):1575–1583
Russ HA et al (2009) Epithelial-mesenchymal transition in cells expanded in vitro from lineage-traced adult human pancreatic beta cells. PLoS One 4(7):e6417
Joglekar MV et al (2009) Human fetal pancreatic insulin-producing cells proliferate in vitro. J Endocrinol 201(1):27–36
Wong WH, Hardikar AA, Joglekar MV (2016) Generation of human islet progenitor cells via epithelial-to-Mesenchymal transition. In: Hardikar AA (ed) Pancreatic islet biology. Springer International Publishing, Cham, pp 217–240
Hardikar AA et al (2003) Human pancreatic precursor cells secrete FGF2 to stimulate clustering into hormone-expressing islet-like cell aggregates. Proc Natl Acad Sci U S A 100(12):7117–7122
Joglekar MV, Hardikar AA (2012) Isolation, expansion, and characterization of human islet-derived progenitor cells. Methods Mol Biol 879:351–366
Wong W et al (2014) Lineage-committed pancreatic progenitors and stem cells. In: Turksen K (ed) Adult Stem Cells. Springer, New York, pp 339–357
Acknowledgments
The support provided to WKMW through the University of Sydney postgraduate awards, MVJ through the Australian Diabetes Society (ADS) Skip Martin fellowship and currently through the JDRF International postdoctoral fellowship, CM through NHMRC clinical Trials Centre, and AAH through the JDRF Australia Career Development Award as well as the visiting professorship through the Danish Diabetes Academy is highly acknowledged.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Maynard, CL., Wong, W.K.M., Hardikar, A.A., Joglekar, M.V. (2019). Droplet Digital PCR for Measuring Absolute Copies of Gene Transcripts in Human Islet-Derived Progenitor Cells. In: Joglekar, M., Hardikar, A. (eds) Progenitor Cells. Methods in Molecular Biology, vol 2029. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9631-5_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9631-5_4
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9630-8
Online ISBN: 978-1-4939-9631-5
eBook Packages: Springer Protocols