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 features articles detailing methods for RNA analysis.

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 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. Like all of our featured articles, these protocols are freely available to subscribers and non-subscribers alike.

RNA-binding proteins play important roles in all aspects of RNA metabolism, particularly in the regulation of mRNAs and subsequent control of gene expression. RNA Immunoprecipitation (RIP), much like Chromatin Immunoprecipitation (ChIP), is a method for analyzing the interactions between proteins and nucleic acids. In the June issue of Cold Spring Harbor Protocols, Jesper Svejstrup and colleagues from the London Research Institute provide RNA Immunoprecipitation to Determine RNA-Protein Associations In Vivo, a detailed set of instructions for RIP analysis. Proteins and RNAs are cross-linked by formaldehyde treatment and immunoprecipitated. RNAs are then recovered and characterized by RT-PCR. The method is particularly useful for kinetic analysis of interactions at different timepoints and under different environmental conditions.

One of our featured articles for February comes from S.P. Dinesh-Kumar and colleagues at Yale University and is an update to a method first published a few years ago in our RNAi manual (now marked way down to a bargain price in both hardcover and paperback). RNAi has become a commonly-used tool for the down-regulation of genes in plants. The most effective means of accomplishing this gene silencing is through the use of viral vectors, with the Tobacco Rattle Virus (TRV) providing the most robust results. Virus-Induced Gene Silencing as a Tool for Delivery of dsRNA into Plants outlines a simple procedure for introducing TRV-based vectors into plants such as Arabidopsis, Nicotiana benthamiana and tomato.

Like all of our featured articles, access to this protocol is free for both subscribers and non-subscribers alike.

Way back in 2003, we published RNAi: A Guide To Gene Silencing, which was one of, if not the first major treatises on the subject. One of the problems with being the first to publish on a fast-moving field is that a book can date quickly. While there’s still much valuable information in RNAi, I’ve been asking authors to update their protocols, which have evolved over the last 5 years or so.

Last month, Esther Stoeckli and colleagues provided an update to her method for Gene Silencing by Injection and Electroporation of dsRNA in Avian Embryos.

This month’s issue brings a tour de force updating and expansion of Petr Svoboda and Paula Stein’s chapter on RNAi in mouse oocytes and early embryos. They’ve written up a general topic introduction on the subject, explanations of how to choose the sequence of dsRNA for RNAi and how to clone and sequence an inverted repeat, and protocols for Cloning a Transgene for Transgenic RNAi in Mouse Oocytes, Preparation of dsRNA for Microinjection, Microinjection of dsRNA into Fully-Grown Mouse Oocytes, Microinjection of dsRNA into Mouse One-Cell Embryos, and Microinjection of Plasmids into Meiotically Incompetent Mouse Oocytes.

Next month will bring an update of Savithramma Dinesh-Kumar’s protocol for using viral vectors for RNAi in plants. More on that in February.

August’s issue of CSH Protocols is now available, and one of the featured protocols this month comes from Inder Verma’s lab, and covers the Design and Cloning of an shRNA into a Lentiviral Vector. Combining the specificity of small interfering RNA (siRNA) silencing with the versatility of lentiviral vectors gives researchers a powerful tool for the investigation of gene function both in vivo and in vitro. There’s also an alternative method available. In the featured method, one undesirable consequence of this procedure is that the siRNA target sequence is also present in the mRNA expressing the marker gene, resulting in somewhat lower expression of the marker. In the alternative method, the position of the silencing cassette is upstream of the marker expression cassette, thus avoiding down-regulation of the marker. But, because the silencing cassette is not in the 3′ LTR, only one copy of the silencing cassette is delivered per viral particle (as opposed to two copies in the featured method).

All of our monthly featured articles are freely available to subscribers and non-subscribers alike.

Those familiar with the CSHL Press Manual, Drosophila Protocols (edited by Sullivan, Ashburner and Hawley) will want to be sure to check CSH Protocols’ February issue, as Bruce Paterson’s group at the National Institutes of Health has written an update of his chapter, “Targeted Disruption of Gene Function in Drosophila by RNA Interference”. The new, up-to-date version of the book chapter appears online in a series of articles, including Preparation of Double-Stranded RNA for Drosophila RNA Interference (RNAi), Collection of Drosophila Embryos for RNA Interference (RNAi), and Injection of dsRNA into Drosophila Embryos for RNA Interference (RNAi) (freely available as one of this month’s featured protocols). Paterson’s group has also contributed a new article covering Drosophila RNA Interference (RNAi) Using a Gal-4 Inducible Transgene Vector.

With the rapid growth of siRNA techniques in so many experimental systems, it’s important to know your options for getting those RNAs into your cells or organism of choice. This month CSH Protocols presents four different methods for delivering siRNAs and shRNAs into various organisms. Read the rest of this entry »