Biochemical and Molecular Biological Assessments of Focal Cerebral Ischemia: mRNA and MicroRNA

Abstract

Evaluation of messenger RNA (mRNA) expression has long been used as evidence demonstrating alterations in gene expression at the transcriptional level following global ischemia. Given the complexity of focal cerebral ischemic injury, in situ analysis of the localization and coexpression of specific RNAs is necessary to tease apart differentially susceptible populations of neurons to ischemic injury. Historically, RNA has been long characterized by its role as the "messenger in the middle" between DNA and proteins, where it was seen as primarily a stopping ground and potential added layer of control of protein expression. However, in recent years, RNA has begun to be understood as much more than simply a messenger. The discovery of microRNA (miRNA) and other small noncoding RNA species has led to a reemergence of RNA-based applications, particularly in the investigation of pathological conditions, such as focal cerebral ischemia. One of the strongest of these techniques lies in the modification of polymerase chain reaction (PCR). In this chapter, we present the basic techniques of reverse transcription-PCR for mRNA and miRNA detection and DIG-labeled oligonucleotide in situ hybridization in the context of focal cerebral injury. © Springer Science+Business Media, LLC 2012.