Methodology Of Northern Blot Analysis

9.1. PREPARATION OF THE RNA FOR ELECTROPHORESIS The method of choice for RNA isolation in many labs is centrifugation through a cesium chloride cushion, followed by ethanol precipitation (22). This method efficiently generates RNA of exceptional quality for Northern blotting. However, alternative methods are available that address limitations of some investigators with respect to instrumentation. The acidic phenol extraction method described by Chomczynski (23) yields total cellular RNA of acceptable quality for Northern blot analysis, can be performed in less time than a cesium chloride gradient, and does not require an ultracentrifuge. Commercial kits are available for the isolation of RNA, most of which are based on variations of acidic phenol extraction.

Regardless of the method utilized to generate total cellular RNA, it is advisable to check the quantity and quality of the samples by taking spectrophotometer readings at 260 and 280 nm and running a small aliquot (1 |g) through a 1.0% agarose integrity gel containing 0.5 |g/mL ethidium bromide. The ratio of the absorbance at 260/280 is 1.8-2.0 in a clean RNA sample. Ratios <1.8 indicate contamination with protein or phenol. Visualization of the integrity in a gel with a UV light source is the best measure of RNA quality (see Fig. 5). DNA contaminants are revealed as very high-molecular-weight bands that sometimes fail to migrate into the gel. Degraded RNA is identified as a smear in the very low-molecular-weight range. When total cellular RNA is analyzed, 1S and 28S rRNA bands should be clearly visible, and a faint smear representing the heterogeneous mRNA population should be present as well. The eukaryotic 28S and 18S rRNAs are 5.0 and 1.87 kb in size, respectively (7), and can serve as a convenient internal RNA size standard.

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