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The probing of RNA gel blots (also called Northern blots) with labeled nucleic acids provides data on the relative levels of steady state gene expression, and on RNA processing. The principle of the technique was first described by Alwine et al. (1). The protocol described in this chapter demonstrates the use of nonradioactive digoxigenin-labeled probes (chapter 10 and chapter 11) on RNA blots (chapter 7 and chapter 8). The detection system employs an antidigoxigenin antibody/alkaline phosphatase conjugate, and the chemiluminescent substrate 3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)-phenyl-1,2-dioxetane (AMPPD) (2,3). The procedure used is similar to that described for probing Southern blots and detection by chemiluminescence in chapter 17. However, this procedure uses a different membrane for support of the immobilized nucleic acids. This, and the fact that RNA is being probed, require modifications to the hybridization buffer and washing procedure. [ABSTRACT FROM AUTHOR]

RNA gel blots (often referred to as Northern gel blots) are frequently used in the analysis of gene expression, for example when investigating specificity of gene expression, quantification of transcription, and analysis of RNA processing. Electrophoresis of RNA through agarose gels for blotting requires complete denaturation of the RNA. A number of denaturants have been used, including glyoxal and dimethyl sulfoxide (1), methylmercuric hydroxide (2), and formaldehyde (3). The following protocol described by Fourney et al. (4), uses formaldehyde as the denaturant but at a lower concentration than previously suggested. This allows direct visualization of the RNA without the need for staining and washing of the gel. The buffer system used is MOPS, which unlike Tris-HCl is suitable for use with formaldehyde see Note 1). The RNA is then transferred to a nylon membrane by capillary blotting for subsequent hybridization. Mol-wt standards can be used to estimate transcript size if required. [ABSTRACT FROM AUTHOR]

Efficient extraction of high quality RNA from a variety of plant tissues is an important first step in many procedures, such as analysis of gene expression, cDNA library construction, and in vitro translation. This procedure, which is essentially as described in Draper et al. (1), involves grinding and phenol extraction of plant material followed by differential precipitation of RNA with sodium acetate. The protocol has been successful with leaf material and cultured cells from a large number of species, however, slight adjustments may be necessary to optimize extraction from other tissues. [ABSTRACT FROM AUTHOR]

RNA dot hybridizations were first described by Kafatos et al. (1). They allow rapid detection of transcription from a number of mRNA populations and are particularly useful in the initial characterization of clones derived from differentially expressed genes. Where accurate quantification of transcription is necessary, or many samples have to be handled, filtration manifold systems are available, such as the Millipore (Bedford, MA) MilliBlot system, that use a vacuum source to transfer nucleic acid to filter. [ABSTRACT FROM AUTHOR]