Bench Marks

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 dynamic nature of biological processes has long been difficult to document, as researchers have been limited to static studies based on fixed specimens. Methods like immunocytochemistry or in situ hybridization can only provide accurate information on one organism at one particular time point. As Scott Fraser has remarked, it’s akin to trying to figure out the rules of football from looking at a set of still photographs taken during a game. But recent developments in imaging techniques, particularly the use of Green Fluorescent Protein (GFP) and its variants, have provided nondestructive ways to study dynamic processes over time, taking our understanding into the fourth dimension.

These new imaging techniques generate an enormous amount of digital image data, which can be difficult to cope with as it builds up over time. Computer-based image analysis is required for the extraction of reproducible and quantitative information. Previously, Cold Spring Harbor Protocols has featured Khuloud Jaqaman and Gaudenz Danuser’s case study using particle tracking to study cellular dynamics. In the June issue of the journal, Roland Eils and colleagues present Tracking and Quantitative Analysis of Dynamic Movements of Cells and Particles. The article sketches a general workflow for quantitative analysis of live cell images and details automated methods for image analysis including preprocessing, segmentation, registration, tracking and classification.

The rapid pace of technological progress in biological imaging has provided great insight into the processes of embryonic development. But for higher organisms with opaque eggs or internal development, optical access to the embryo is limited. While various embryonic culture methods are available, vertebrate development is best studied in an intact embryo model, one in which the natural environment has not been disrupted. In the June issue of Cold Spring Harbor Protocols, Paul Kulesa and colleagues from the Stowers Institute for Medical Research present In Ovo Live Imaging of Avian Embryos, a detailed set of instructions for time-lapse imaging of fluorescently labeled cells within a living avian embryo. During the procedure, a hole is made in the shell, and a Teflon membrane that is oxygen-permeable and liquid-impermeable is used to provide a window for visualization of the embryo via confocal or two-photon microscopy. Imaging can take place for up to five days without dehydration or degradation of the normal developmental environment. As one of June’s featured articles, the protocol is freely available to subscribers and nonsubscribers alike. Kulesa’s group also supplies a second protocol in the issue, covering Multi-Position Photoactivation and Multi-Time Acquisition for Large-Scale Cell Tracing in Avian Embryos, a technique that produced June’s cover image.