Isolation of RNA and the Synthesis and Amplification of cDNA from Antigen-Specific T Cells for Genome-Wide Expression Analysis

Abstract

Genome-wide gene expression analysis has become a very powerful routine tool for the study of distinct differentiation states. However, the examination of total populations of cells that contain high levels of heterogeneity, such as the total CD8⁺ T cell population during an immune response, is limited because that complexity hampers accurate interpretation. The gene expression signatures from populations represent the average of all cells within the populations, which will smooth out large expression changes within small subpopulations and virtually eliminate any small changes. However, small expression changes within a minor subpopulation, such as antigen-specific CD8⁺ T cells responding to an infection, can have relevant biological consequences. Although very limited amounts of RNA can be isolated from small subpopulations of cells, there are now methods to synthesize and amplify cDNA from this limited RNA in sufficient quantities needed for microarray analysis. Here, we describe a complete protocol to extract RNA from small numbers of cells, synthesize cDNA from that RNA, and amplify that cDNA in an unbiased method. This protocol is a useful tool for the study of genome-wide expression signatures from many of the subpopulations that are numerically small but important in immune responses and homeostasis.