Use of real-time polymerase chain reaction to measure gene expression in single cells.
Quantification of the expression of individual genes can reveal much concerning the processes occurring within a cell. In the vast majority of cases, activation or repression of a gene is indicative of altered utilization of the pathway or process in which it functions. Although microarray analysis has the power to provide data concerning the expression of thousands of genes in a single experiment, validation of results using alternative methods is still essential. This is particularly true if the amount of RNA available for microarray analysis is very small, necessitating methods of RNA amplification. The gold-standard for quantifying mRNA transcripts from an individual gene is the use of reverse transcription followed by real-time PCR. This approach has yielded highly accurate and reproducible data, even when applied to minute samples, such as single oocytes or single embryos. This chapter describes protocols for the quanitification of mRNA transcripts using real-time PCR and considers issues specific to analysis of single cells.