An improved one-tube RT-PCR protocol for analyzing single-cell gene expression in individual mammalian cells.
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.
Li
Yongzhong
Y
Center for Ecogenomics, The Biodesign Institute, Arizona State University, P.O. Box 876501, Tempe, AZ 85287-6501, USA. klee0127@gmail.com
Thompson
Hansa
H
Hemphill
Courtney
C
Hong
Fan
F
Forrester
Jessica
J
Johnson
Roger H
RH
Zhang
Weiwen
W
Meldrum
Deirdre R
DR
D045744:Cell Line, Tumor; D002477:Cells / Q000737:chemistry* / Q000166:cytology; D015870:Gene Expression; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*