Chapter 25 - A Few RNA Success Stories

Abstract

The protocols and accompanying discussion in the preceding chapters of this text illustrate various methods for the expedient isolation of RNA from a variety of biological sources and its subsequent analysis. The judicious application of various combinations of these techniques constitutes a systematic method for understanding the role of the products of transcription in the regulation of gene expression. Any experimental model can be well-characterized by assaying specific mRNAs as one parameter of gene expression, although having protein expression data from Western analysis, immunochemical, or other proteomics techniques generally provides a more complete understanding of gene regulation under a specific set of conditions. A typical RNA-based experimental design includes an assessment of steady-state levels of RNA, the levels of related regulatory molecules such as miRNAs, and how these elements change in response to naturally occurring variance or experimental challenge. Transcription rate data can provide additional insight pertaining to the regulatory profile of a gene when examined along with data from steady-state transcription assays. Because experiments involving RNA are almost certainly asking gene expression questions, these techniques are commonly performed to assign observed changes to the transcriptional level or due to some posttranscriptional event(s). Therefore, assays providing the greatest sensitivity and resolution are always desirable. Real-time PCR, miRNA cataloging, and innovations in sequencing, particular RNA-Seq, have facilitated profiling the depths of the cellular biochemistry at levels inconceivable fifteen years ago and these methods continue to offer great potential. Bioinformatics, email, and all types of online tools provide a platform for literature search or a great deal of background reading without leaving one’s desk. There are a number of different Internet-based bulletin boards where scientists with difficulties can post specific questions and hope for an answer from the readership.