Ž . Mutation Research Genomics 406 1999 45–54 www.elsevier.comrlocatermrgenomics Community address: www.elsevier.comrlocatermutres Gene expression in individual cells: analysis using global single cell reverse transcription polymerase chain reaction ž / GSC RT-PCR Leslie H. Brail a,b , Anne Jang b , Filio Billia a,c , Norman N. Iscove a,c , Henry J. Klamut a,b , Richard P. Hill a,b, ) a Department of Medical Biophysics, UniÕersity of Toronto, Toronto, Ontario, Canada, M5G 2M9 b DiÕision of Experimental Therapeutics, Ontario Cancer Institute r Princess Margaret Hospital, 610 UniÕersity AÕenue, Toronto, Ontario, Canada, M5G 2M9 c DiÕision of Cell and Molecular Biology, Ontario Cancer Institute r Princess Margaret Hospital, 610 UniÕersity AÕenue, Toronto, Ontario, Canada, M5G 2M9 Accepted 4 November 1998 Abstract 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 Ž . 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 Ž 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. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Gene expression; Cell; GSC RT-PCR ) Corresponding author. Division of Experimental Therapeutics, Ontario Cancer InstituterPrincess Margaret Hospital, 610 Univer- sity Avenue, Toronto, Ontario, Canada, M5G 2M9; Tel.: q1-416- 946-2979; Fax.: q1-416-946-2984; E-mail: hill@oci.utoronto.ca 1. Introduction Many aspects of both normal development and disease result from changes in the expression of wx multiple genes in individual cells 1 . It is widely Ž accepted that cancer is a clonal disease i.e., it arises . from a single cell and that tumor progression occurs 1383-5734r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S1383-5726 98 00009-0