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An improved one-tube RT-PCR protocol for analyzing single-cell gene expression in individual mammalian cells. Anal Bioanal Chem 2010 Jul;397(5):1853-9

Date

05/22/2010

Pubmed ID

20490471

DOI

10.1007/s00216-010-3754-0

Scopus ID

2-s2.0-77953811012   14 Citations

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 microl 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.

Author List

Li Y, Thompson H, Hemphill C, Hong F, Forrester J, Johnson RH, Zhang W, Meldrum DR

Author

Roger H. Johnson PhD Associate Professor in the Biophysics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Cell Line, Tumor
Cells
Gene Expression
Humans
Reverse Transcriptase Polymerase Chain Reaction