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TaqMan® OpenArray® High-Throughput Transcriptional Analysis of Human Embryonic and Induced Pluripotent Stem Cells

  • Sunali N. Patel
  • Yalei Wu
  • Yun Bao
  • Ricardo Mancebo
  • Janice Au-Young
  • Elena Grigorenko
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 997)

Abstract

It is widely accepted that somatic cells can be reprogrammed by a set of transcription factors to become embryonic stem cell-like: These reprogrammed cells, induced pluripotent stem cells (iPSCs), are nearly identical to embryonic stem cells (ESCs), because both have the capacity to self-renew and to form all cellular lineages of the body. Transcriptional differences between ESCs, iPSCs, and fibroblasts can be analyzed by quantitative PCR (qPCR) using TaqMan® Gene Expression assays, a widely used tool for rapid analysis of different cell types. In this chapter, we describe the OpenArray® platform which generates qPCR data from high-throughput instrumentation. We examined the gene signature profiles of ESCs, fibroblasts, and iPSCs with a TaqMan® OpenArray® Human Stem Cell Panel containing 631 TaqMan® Gene Expression assays that represent pathways involved in self-renewal, pluripotency, lineage patterning, transcriptional networks, stem cell differentiation, and development.

Key words

Reprogramming Fibroblast Induced pluripotent stem cells iPSC TaqMan RT-PCR Pluripotency Self-renewal Embryonic stem cells Adult stem cells 

Notes

Acknowledgements

The authors thank Rene Quintanilla and Uma Lakshmipathy for providing the cells used in this study, Doug Roberts for assay selection, Vanee Pho for technical guidance and documentation, and Caifu Chen and David Keys for support and helpful discussions.

References

  1. 1.
    Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676PubMedCrossRefGoogle Scholar
  2. 2.
    Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M (2009) Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci 85:348–362PubMedCrossRefGoogle Scholar
  3. 3.
    Seki T, Yuasa S, Oda M, Egashira T, Yae K, Kusumoto D, Nakata H, Tohyama S, Hashimoto H, Kodaira M, Okada Y, Seimiya H, Fusaki N, Hasegawa M, Fukuda K (2010) Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells. Cell Stem Cell 7:11–14PubMedCrossRefGoogle Scholar
  4. 4.
    Zhang X, De Los Angeles A, Zhang J (2010) The art of human induced pluripotent stem cells: the past, the present and the future. Open Stem Cell J 2:2–7Google Scholar
  5. 5.
    Morrison T, Hurley J, Garcia J, Yoder K, Katz A, Roberts D, Cho J, Kanigan T, Ilyin SE, Horowitz D, Dixon JM, Brenan C (2006) Nanoliter high throughput quantitative PCR. Nucleic Acids Res 34(18):e123PubMedCrossRefGoogle Scholar
  6. 6.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-delta delta C(T)) method. Methods 25(4):402–408PubMedCrossRefGoogle Scholar
  7. 7.
    Adewumi O, Aflatoonian B, Ahrlund-Richter L et al (2007) Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat Biotechnol 25(7):803–816PubMedCrossRefGoogle Scholar
  8. 8.
    Mestdagh P, Van Vlierberghe P, De Weer A, Muth O, Westermann F, Speleman F, Vandesompele J (2009) A novel and universal method for miRNA RT-qPCR data normalization. Genome Biol 10:R64PubMedCrossRefGoogle Scholar
  9. 9.
    Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of quantitative RT-PCR data by geometric averaging of multiple internal genes. Genome Biol 3:0034-1–0034-11CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Sunali N. Patel
    • 1
  • Yalei Wu
    • 1
  • Yun Bao
    • 1
  • Ricardo Mancebo
    • 1
  • Janice Au-Young
    • 1
  • Elena Grigorenko
    • 1
  1. 1.Genetic AnalysisLife TechnologiesForter CityUSA

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