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An improved one-tube RT-PCR protocol for analyzing single-cell gene expression in individual mammalian cells

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Abstract

It is well known that gene expression is regulated at the level of individual cells, and more evidence is now emerging that heterogeneity of cell regulation is orders of magnitude greater than previously thought. In order to detect meaningful variations in transcription levels, it is necessary to measure gene expression at single cell levels rather than in bulk cells, where individual differences or heterogeneity could be lost. In this work, we report an improved reverse-transcriptase polymerase chain reaction (RT-PCR) protocol which allows the direct measurement of gene expression in one tube (5–25 μl of total PCR mixture) at the single mammalian cell level. The protocol employs a new cell lysis buffer, and involves no RNA isolation or nested PCR steps, significantly reducing the possibility of contamination and errors. We successfully applied this protocol in qRT-PCR and linear-after-the-exponential (LATE)-PCR to analyze selected genes of various expression levels from three cell lines. Although further characterization of RNA stability is important, the preliminary results showed that gene expression heterogeneity could be common among members of genetically identical cell populations. The protocol illustrated can be utilized for a wide array of applications without much modification, such as cancer cell analysis and preimplantation genetic diagnostics. In addition, the protocol is based on intercalator-based (SYBR Green PCR) chemistry, which is less expensive and suitable for high-throughput platforms.

Gene expression heterogeneity in genetically identical cell population. qRT-PCR analysis of p53 transcript in HeLa cells with two negative controls in red. (A) qRT-PCR analysis of p53 transcript with 10 cells in each PCR. 10 PCR were analyzed; (B) qRT-PCR analysis of p53 transcript with 5 cells in each reaction. 8 PCR were analyzed; (C) qRT-PCR analysis of p53 transcript with single cell in each reaction tube. 9 reactions were analyzed; and (D) Statistical analysis on Ct values from panel A, B, and C above. The final total volume is 25 μl for all PCR analysis.

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Acknowledgements

We would like to thank Drs. L. Wangh and A. Sanchez from Brandies University for their help with LATE-PCR technology. We would also like to thank Dr. B. Reid for kindly supplying the CP-C cells. Dr. Y. Anis is acknowledged for his help with single-cell loading. This work was supported by a CEGS Microscale Life Science Center (MLSC) project (Grant #: p50-HG002360) from NIH, USA.

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Authors and Affiliations

  1. Center for Ecogenomics, The Biodesign Institute, Arizona State University, P.O. Box 876501, Tempe, AZ, 85287-6501, USA

    Yongzhong Li, Hansa Thompson, Courtney Hemphill, Fan Hong, Jessica Forrester, Roger H. Johnson, Weiwen Zhang & Deirdre R. Meldrum

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  1. Yongzhong Li
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  2. Hansa Thompson
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  3. Courtney Hemphill
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  4. Fan Hong
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  5. Jessica Forrester
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  6. Roger H. Johnson
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  7. Weiwen Zhang
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  8. Deirdre R. Meldrum
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Correspondence to Yongzhong Li or Weiwen Zhang.

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Li, Y., Thompson, H., Hemphill, C. et al. An improved one-tube RT-PCR protocol for analyzing single-cell gene expression in individual mammalian cells. Anal Bioanal Chem 397, 1853–1859 (2010). https://doi.org/10.1007/s00216-010-3754-0

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  • DOI: https://doi.org/10.1007/s00216-010-3754-0

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