Romero-López Cristina, Barroso-delJesus Alicia, Menendez Pablo, Berzal-Herranz Alfredo
Instituto de Parasitología y Biomedicina López-Neyra, IPBLN-CSIC, Parque Tecnológico de Ciencias de la Salud, Av del Conocimiento s/n, Granada, Spain.
Methods Mol Biol. 2012;809:491-503. doi: 10.1007/978-1-61779-376-9_32.
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.
基因表达是一个多步骤过程，从DNA经RNA到蛋白质。该过程的严格调控对于整体细胞完整性和生理稳态至关重要。信使RNA（mRNA）水平的调控已成为调节遗传信息表达的关键事件。这一过程发生的机制已得到广泛研究，并且开始被更好地理解。它们涉及一个复杂的途径网络，利用靶mRNA的内在特征，如稳定性，来控制其在细胞质中的相对丰度。因此，分析mRNA的稳定性和丰度对于正确开展基因表达研究至关重要。本章介绍核糖核酸酶保护分析，这是一种广泛接受的评估靶mRNA质量和数量的方法。该技术比传统的Northern印迹分析具有更高的灵敏度，可单独使用，也可与其他定量方法（如定量逆转录PCR）结合使用，作为互补程序，提供关于基因表达更完整、更可靠的信息。
