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Microsatellites pp 147–166Cite as

Development of a Multiplex PCR Assay for Characterization of Embryonic Stem Cells

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1006))

Abstract

Several molecular methods like real-time PCR (Q-PCR), expression sequence tag (EST) scan, microarray and microRNA analysis, and massively parallel signature sequencing (MPSS) have proved to be increasingly sensitive and efficient for monitoring human embryonic stem cell (hESC) differentiation. However, most of these high-throughput tests have a limited use due to high cost, extended turnaround time, and the involvement of highly specialized technical expertise. Hence, there is a need of rapid, cost-effective, robust, yet sensitive method for routine screening of hESCs. A critical requirement in hESC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of germ-layer-specific gene markers. To determine the modulation of gene expression in hESCs during propagation, expansion, and differentiation via embryoid body (EB) formation, we developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR (mxPCR) platform technology. Among the 15 gene primers tested, 4 were pluripotent markers comprising of set 1; and 3 lineage-specific markers from each ecto-, meso-, and endoderm layers were combined as sets 2, 3, and 4, respectively. In summary, this study was performed to characterize hESCs on a molecular level and to determine the quality and degree of variability among hESC and their early progenies (EB). This single-reaction mxPCR assay was flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC lines during routine maintenance and directed differentiation.

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Acknowledgment

The authors are grateful to Stempeutics Research Malaysia for facilitating our research on stem cells and regenerative medicine.

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Authors and Affiliations

  1. Manipal Institute of Regenerative Medicine, Manipal University, Bangalore, India

    Rajarshi Pal, Murali Krishna Mamidi & Ramesh Bhonde

  2. Stem Cells and Regenerative Medicine, Stempeutics Research Malaysia Sdn Bhd, Kuala Lumpur, Malaysia

    Anjan Kumar Das

  3. NIH Center for Regenerative Medicine, National Institutes of Health, Bethesda, MD, USA

    Mahendra Rao

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  1. Rajarshi Pal
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  2. Murali Krishna Mamidi
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  4. Mahendra Rao
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Editors and Affiliations

  1. , Department of Plant, Soil,, Southern Illinois University, Carbondale, Lincoln Drive 1205, Carbondale, 62901, Illinois, USA

    Stella K. Kantartzi

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Pal, R., Mamidi, M.K., Das, A.K., Rao, M., Bhonde, R. (2013). Development of a Multiplex PCR Assay for Characterization of Embryonic Stem Cells. In: Kantartzi, S. (eds) Microsatellites. Methods in Molecular Biology, vol 1006. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-389-3_11

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  • DOI: https://doi.org/10.1007/978-1-62703-389-3_11

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-388-6

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