SINGLE STEP PROKARYOTIC RNA ISOLATION

In the past, poly(A)+ RNA has not been detected in prokaryotes.  In the early 80s, a new method of isolating RNA from bacteria was developed, involving lysis by protease K in the presence of SDS and potent RNase inhibitors (heparin & 1,10 phenanthroline) and without phenol:chloroform extractions.  This method has allowed the detection of poly(A)+ RNA in a variety of prokaryotes: Bacillus subtilus, B. brevis, B. polymyxa, E. coli, Rhodospirillum rubrum, Rhodopseudomonas capsulata, Caulobacter cresentus, Hypomicrobium B-522, Anabena variabilis and Nostoc MAC.  It is possible that the phenol:chloroform extractions used in earlier procedures selectively eliminates poly(A)+ RNA or mRNA , although this is not the case when isolating RNA from eukaryotes.  RNA s prepared by this method are very crude, but can be used directly for oligo(dT)-cellulose chromatography or urea-agarose gel electrophoresis, or can be further purified by any method NOT including organic extraction.

RNase-free technique should be used throughout this procedure, in spite of the RNase inhibitors.

Lysis buffer (per 200ml)      final conc.     working conc.

1.9380 grams Tris, pH 7.5      80 mM          35.6 mM

0.4067 grams MgCl₂-7H₂O        10 mM           4.4 mM

140 μl 2-mercaptoethanol        10 mM           4.4 mM

5% SDS - 1 gram per 20ml

2.3 mg/ml Protease K - 46 mg in 20ml ddH₂O

0.1M EDTA - 4ml 0.5M EDTA + 16ml ddH₂O

20mM 1,10-phenanthroline - 79 mg in 20ml ddH₂O(boil to dissolve)

2 mg/ml Heparin - 40 mg in 20ml ddH₂O

2.5M Na acetate - 68 grams per 200ml

Filter sterilize protease K, 1,10-phenanthroline and heparin.

1. Pellet 1.5ml stationary phase or 3ml log phase cells in a microfuge tube.  Decant the supernatant and remove residual droplets from the tube with a pasteur pipette.

2. Resuspend the pellet in 200ul lysis buffer.  Add 45ul each of the following in sequence:

2 mg/ml heparin

20mM 1,10-phenanthroline

0.1M EDTA

5% SDS

2.3 mg/ml protease K

3. Vortex and incubate 20 min. at 37℃.  (Some species may require sonication prior to proteolysis to lyse the cells, i.e. Bacillus)

4. Add 50ul 2.5M Na acetate and 1ml EtOH.  Vortex, and incubate ON -20℃.

5. Pellet 15 min. and resuspend in 20ul AN.  Store at -20℃.

NOTE: For oligo(dT)-cellulose chromatography, omit steps 4 & 5, but add 50μl 5M NaCl and chromatograph immediately, or freeze on dry ice and store at -70 for future chromatography.