Evaluation of RNA Quality

1. Quantitate the RNA by spectrophotometry. OD260:280 ratios should be 1.8 or greater, although tissues containing significant quantities of proteoglycans may give lower ratios. If the ratio is too low, phenol-extract, chloroform-extract, and ethanol-precipitate the RNA one or more times.

2. Evaluate the integrity of the RNA by gel electrophoresis, either denaturing or nondenaturing.

3. Nondenaturing agarose gels: The simplest method is to run a 0.8% agarose gel containing 100 pg/mL ethidium bromide (EtBr), using TBE as the running buffer, just as for a normal DNA gel.

4. Denaturing gel—use a 0.8% formaldehyde-agarose gel run in MOPS-acetate buffer as described in ref. (9). Stain with ethidium bromide for 30', and view under UV.

5. The 28S and 18S rRNA bands should be sharp and in a 2:1 ratio. Some organisms, e.g., Drosophila melanogaster and Alligator mississipiensis, have 28S rR-NAs, which are nicked and migrate with the 18S bands in denaturing gels. Use nondenaturing gels with these organisms and if you do not see the 28S band in denaturing gels.

6. Storage of RNA—store at -70°C in H2O or as an ethanol precipitate at -20°C (preferred).

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