Quantitative analysis of gene transcription in stroke models using real-time RT-PCR

Many researchers have sought to study changes in gene expression in preclinical models of stroke. These range from in vitro models of ischemia, neuronal death, and regeneration to in vivo animal models aimed at replicating pathologies and regenerative processes typical of the clinical situation. In all such models, changes in gene expression occur, which may be assessed by measuring the abundance of the mRNA transcribed from particular genes of interest. The advent of real-time reverse-transcriptase polymerase chain reaction (RT-PCR) has vastly improved the sensitivity and accuracy of mRNA detection and is now the method of choice in many studies. Although this is a relatively simple and rapid technique, it has a number of pitfalls, especially in experimental design and data analysis. In this chapter we describe a detailed experimental protocol for real-time RT-PCR detection of mRNA transcripts, as used in the rat permanent middle cerebral artery occlusion model. We also discuss methods for analysis and interpretation of the resulting data.