119 – Practical Methods for Determining mRNA Levels in Alcohol-Exposed Tissues and Its Application to Experimental Pathology

This chapter focuses on the practical methods for the determination of Messenger Ribonucleic Acid (mRNA) levels in the alcohol exposed tissues and application to pathology. The exposure of tissues to alcohol is often accompanied by changes in mRNA levels, either as a compensatory adaptation or pathophysiological reaction. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is a useful technique for detecting the low-abundance mRNA, but its quantitative application is limited. The following chapter describes semiquantitative RT-PCR methods developed for evaluating gene expression in a variety of diseases and metabolic conditions, including alcoholism. This method is based on the co-amplification of the target mRNA [e.g. mRNA encoding heat-shock proteins (HSPs) or antioxidants] with an endogenous mRNA as an internal control (e.g. mRNA encoding glyceraldehyde-3-phosphate-dehydrogenase or β-actin). The quantity of the target mRNA is expressed relative to the internal control. To avoid the influence of a large amount of control gene on the amplification efficiency of the target gene, the amount of polymerase chain reaction product of the control gene is reduced in two ways: (1) by limiting the concentration of control primers, and secondly, and (2) by delaying the addition of control primers until several cycles of amplification of the target have been completed.