Gene expression in individual cells: analysis using global single cell reverse transcription polymerase chain reaction (GSC RT-PCR).
The determination of the gene expression pattern of single cells has important implications for many areas of cellular and developmental biology including lineage determination, identification of primitive stem cells and temporal gene expression patterns induced by changes in the cellular microenvironment. Global Single Cell Reverse Transcription-Polymerase Chain Reaction (GSC RT-PCR) enables the study of single cell gene expression patterns. Initial observations of significant heterogeneity among single cells derived from a population of cells prompted us to determine how much of this observed heterogeneity was due to the intrinsic variation within the method. In this paper we discuss the sensitivity of GSC RT-PCR for analysis of differences in gene expression between single cells and, in particular, detail the amount of variation generated by the method itself. We found that most of the intrinsic variation in the method occurred in the PCR step. The total variation induced by the method was in the range of 5 fold. While we have determined that there is a five fold methodological variation in GSC RT-PCR, any method which use its components (including generation of cDNAs for microarray analysis) is likely to be affected by such experimental variability, which could limit the interpretation of the resulting data.
Brail
L H
LH
Department of Medical Biophysics, University of Toronto, Toronto, Canada.
Jang
A
A
Billia
F
F
Iscove
N N
NN
Klamut
H J
HJ
Hill
R P
RP
D000704:Analysis of Variance; D000818:Animals; D002457:Cell Extracts / Q000235:genetics; D002460:Cell Line; D015342:DNA Probes; D018076:DNA, Complementary / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D020559:Monomeric GTP-Binding Proteins; D054778:NM23 Nucleoside Diphosphate Kinases; D009701:Nucleoside-Diphosphate Kinase; D012333:RNA, Messenger / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics; D055293:Receptors, Urokinase Plasminogen Activator; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012269:Ribosomal Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000378:metabolism
D002457:Cell Extracts; D015342:DNA Probes; D018076:DNA, Complementary; D054778:NM23 Nucleoside Diphosphate Kinases; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D012269:Ribosomal Proteins; D014157:Transcription Factors; C041806:ribosomal protein L32; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D009701:Nucleoside-Diphosphate Kinase; D020559:Monomeric GTP-Binding Proteins