Preparation of RNA Gel Blots

Abstract

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.