Appendix A - Experimental protocols

Summary

Total RNA isolation from bacteria

Total RNA is isolated from cells at an A₆₀₀ of 0.5–0.6. Ten-ml samples of cultures of growing cells are pipetted directly into 10 ml of boiling lysis buffer (1% SDS, 0.1 m NaCl, 8 mm EDTA) and mixed at 100°C for 1.5 min. These samples are transferred to 250-ml Erlenmeyer flasks, mixed with an equal volume of acid phenol (pH 4.3), and shaken vigorously for 6 min at 64°C. After centrifugation, the aqueous phase is transferred to a fresh Erlenmeyer flask, and the hot acid phenol extraction procedure is repeated. The second aqueous phase is extracted with phenol:chloroform:isoamyl alcohol (25:24:1, pH 4.3) at room temperature and, finally, twice with chloroform-isoamyl alcohol (24:1). Total RNA is precipitated with two volumes of ethanol in 0.3 m sodium acetate (pH 5.3), washed with 70% ethanol, and redissolved in RNAase-free water. The contaminating genomic DNA is removed from the total RNA with Ambion's DNA-free kit ™ (catalog no. 1906). Since genomic DNA is a common source of high background on DNA arrays, this step is repeated at least once. The quality and integrity of the total RNA preparation is ascertained by electrophoresis in a 1.2 % agarose gel run in 1 × TAE (40 mm Tris-acetate, 2 mm EDTA) buffer. Appropriate modifications of these methods can be used for total RNA extraction from eukaryotic cells.