Single-cell gene expression profiling using reverse transcription quantitative real-time PCR

Abstract

Even in an apparently homogeneous population of cells there are considerable differences between individual cells. A response to a stimulus of a cell population or tissue may be consistent and gradual while the single-cell response might be binary and apparently irregular. The origin of this variability may be preprogrammed or stochastic and a study of this phenomenon will require quantitative measurements of individual cells. Here, we describe a method to collect dispersed single cells either by glass capillaries or flow cytometry, followed by quantitative mRNA profiling using reverse transcription and real-time PCR. We present a single cell lysis protocol and optimized priming conditions for reverse transcription. The large cell-to-cell variability in single-cell gene expression measurements excludes it from standard data analysis. Correlation studies can be used to find common regulatory elements that are indistinguishable at the population level. Single-cell gene expression profiling has the potential to become common practice in many laboratories and a powerful research tool for deeper understanding of molecular mechanisms.