Development of a multiplex PCR assay for characterization of embryonic stem cells

Pal, Rajarshi and Mamidi, Murali Krishna and Bhonde, Ramesh (2013) Development of a multiplex PCR assay for characterization of embryonic stem cells. Methods in Molecular Biology, 1006. pp. 147-166.

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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 ef fi cient 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-speci fi c 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 de fi nitive 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-speci fi c 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 singlereaction mxPCR assay was fl exible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC lines during routine maintenance and directed differentiation.

Item Type: Article
Uncontrolled Keywords: Human embryonic stem cells; Embryoid body ; Multiplex PCR ; Molecular characterization ; Pluripotency ; Differentiation.
Subjects: Regenerative Medicine > MIRM Bangalore
Depositing User: MCON Library
Date Deposited: 10 Oct 2015 09:54
Last Modified: 10 Oct 2015 09:54

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