RNA Interference (RNAi)/siRNA

The zebrafish (Danio rerio) has rapidly become a favored model organism for studying developmental biology. One of the most commonly used methods for genetic manipulation in the zebrafish is the delivery of plasmids or oligonucleotides to cells within the living embryo via electroporation. When cells are exposed to brief electrical fields, transient membrane destabilization occurs and nucleic acids can cross the plasma membrane. When the electrical field is removed, the membrane seals and the nucleic acids are trapped inside the cell. In vivo electroporation has proven particularly effective for delivering fluorescent protein expression vectors for imaging and loss-of-function reagents such as morpholinos or RNA interference (RNAi) constructs for the knockdown of gene function. In the July issue of Cold Spring Harbor Protocols, Jack Horne and colleagues present Targeting the Zebrafish Optic Tectum Using In Vivo Electroporation, a modification of the technique that can be used to specifically target the developing optic tectum, the midbrain’s visual processing center. Instructions are given for the construction of electroporation electrodes, preparation and injection of DNA, and electroporation of the DNA into the embryonic brain.

The June issue of Cold Spring Harbor Protocols includes an early preview of CSHL Press’ forthcoming RNA: A Laboratory Manual. Protocols covering basic RNA techniques are now available, including methods for purification of RNA by by SDS Solubilization and Phenol Extraction and by Using TRIzol (TRI Reagent), Ethanol Precipitation of RNA and the Use of Carriers, Preparation of Cytoplasmic and Nuclear RNA from Tissue Culture Cells, Removal of Ribosomal Subunits (and rRNA) from Cytoplasmic Extracts before Solubilization with SDS and Deproteinization, Removal of DNA from RNA, Nondenaturing Agarose Gel Electrophoresis of RNA and Polyacrylamide Gel Electrophoresis of RNA.

The last two on that list cover gel electrophoresis, two of the most important and frequently used techniques in RNA analysis. Electrophoresis is used for RNA detection, quantification, purification by size and quality assessment. Gels are involved in a wide variety of methods including northern blotting, primer extension, footprinting and analyzing processing reactions. The two most common types of gels are polyacrylamide and agarose. Polyacrylamide gels are used in most applications and are appropriate for RNAs smaller than approximately 600 nucleotides (agarose gels are preferred for larger RNAs). Polyacrylamide Gel Electrophoresis of RNA describes how to prepare, load and run polyacrylamide gels for RNA analysis. The is featured in the June issue, and as one of our featured articles, the full-text version is available to subscribers and non-subscribers alike.

This set is just a small sampling of the manual’s contents, basic techniques from an early chapter. The full table of contents can be seen here.

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.

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