COLD SPRING HARBOR, N.Y. (Thurs., Oct. 1, 2009) – The study of RNA has long been the tool of choice for understanding where and when genes are expressed in a cell, tissue, or organism during development or under specific physiological or environmental conditions. Recent discoveries have revolutionized our concept of RNA function; it is now known to be active in a much wider set of biological processes than was previously believed. Techniques for isolating RNA and for uncovering its interactions with proteins have taken on new importance as many laboratories define the roles of specific RNAs in the cell. The October issue of Cold Spring Harbor Protocols (www.cshprotocols.org/TOCs/toc10_09.dtl) features two articles detailing methods for RNA analysis in zebrafish and the worm C. elegans.
Quantitative Real-Time RT-PCR (qRT-PCR) of Zebrafish Transcripts: Optimization of RNA Extraction, Quality Control Considerations and Data Analysis from Donald Love and colleagues at the University of Auckland (http://www.sbs.auckland.ac.nz/uoa/science/about/departments/sbs/research/molecular-cellular/love-don/associate-professor-don-love.cfm) presents an optimized method for RNA isolation from zebrafish, along with quality assessment and the use of reference genes. A protocol for quantitative real-time polymerase chain reaction (qRT-PCR) is also included. The article is freely available on the website for Cold Spring Harbor Protocols (http://cshprotocols.cshlp.org/cgi/content/full/2009/10/pdb.prot5314).
RNA molecules interact with proteins to drive many cellular activities, including post-transcriptional processing of RNA, regulation of translation, and transport of RNA to name but a few. These ribonucleoprotein complexes are isolated by coimmunoprecipitation (co-IP), where a protein-specific antibody is used to purify the protein of choice and its associated complex members. Analysis of RNA-Protein Complexes by RNA Coimmunoprecipitation and RT-PCR Analysis from Caenorhabditis elegans gives step-by-step instructions for RNA co-IP from C. elegans whole-worm extracts. The protocol, from Christian Eckmann and colleagues at the Max Planck Institute of Molecular Cell Biology and Genetics (http://www.mpi-cbg.de/research/research-groups/christian-eckmann.html), starts with the large-scale growth of worms and describes the preparation of whole-worm extracts, RNA co-IP, isolation of the purified RNA, and identification of specific genes through RT-PCR. The article is freely accessible on the website for Cold Spring Harbor Protocols (http://cshprotocols.cshlp.org/cgi/content/full/2009/10/pdb.prot5300).
Source: Cold Spring Harbor Laboratory