Analysis of mRNA abundance and stability by ribonuclease protection assay

Abstract

Gene expression is a multi-step process, which proceeds from DNA through RNA to protein. The tight regulation of this process is essential for overall cellular integrity and physiological homeostasis. Regulation of the messenger RNA (mRNA) levels has emerged as a crucial event in the modulation of the expression of genetic information. The mechanisms by which this process occurs have been extensively studied and begin to be much better understood. They involve a network of complex pathways that use intrinsic features of the target mRNA, like stability, to control its relative abundance in the cytoplasm. Thus, the analysis of the mRNA stability and abundance is essential to properly undertake gene expression studies. This chapter describes the ribonuclease protection assay, a widely accepted approach to evaluate the quality and amount of a target mRNA. This technique displays a higher sensitivity than classical Northern blot analysis and may be used either individually or in combination with other quantitative methods, such as quantitative reverse-transcription PCR, as complementary procedures rendering more complete and reliable information on gene expression.