Rapid mini-preparations of total RNA from bacteria

While a wide variety of methods exist for RNA preparation from different cell types/organisms, we describe a small scale preparative method that to date has worked for over 10 different bacterial genera. This method is a modification of a large scale method described for B.subtilis (1). We developed this method so that it would fuilfil several criteria required for work in this laboratory on detection of gene expression of bacterial pathogens in environmental samples. These criteria are: simplicity, small scale (start off volumes of less than 1.5 ml and 108 cells), no requirement for physical contact of the sample with equipment which could become a source of cross-contamination on multisample analysis and finally the production of nuclease-free intact total RNA suitable for analysis by gel electrophoresis, Northern blotting and RNA-targeted amplification reactions. The protocol is: pellet cells in an Eppendorf microfuge at 8000 g for 2 min. Resuspend cell pellet in 20 yl of DEPC treated H20 (2) and add 3 11 0.5% v/v DEPC (diluted in H20). Add 200 ,ul ice-cold acetone, mix by hand and centrifuge at 10,000 g for 2 min. Remove supernatent with care, resuspend pellet in 30 j1l of DEPC-H20 and add 1 tl 100 /Ag/ml Proteinase K. Incubate on ice for 10 min. Add 3.5 tdl DEPC (0.5% v/v), 200 t1l phenol (preheated to 70°C), 150 yl chloroform, mix by hand, add a further 120 IlI DEPC-H20 and centrifuge at 12,000 g for 5 min. Remove the aqueous phase (top layer) and add to 1 ml ethanol. Allow RNA to precipitate for 1 hr at -20°C. Pellet RNA at 12,000 g for 15 min, resuspend in 50-100 IL DEPC-H20 and store at -20°C. Figure 1 shows the RNA products prepared from 106 cells of different bacterial strains electrophoresed on 1.2% TBE agarose gels. Intact 16S and 23S rRNA bands can be visualised. Figure 2 shows the result of a Northern blot on total RNA prepared from E. coli and hybridised with oligodeoxynucleotides derived from the E. coli 16S rRNA and 4.5S RNA genes. Discreet bands show that the prepared RNA is intact and as such a suitable template for Northern blot analysis. Contaminating genomic DNA has been found in some preparations, particularly from Gram + bacteria, but due to its large molecular weight has no deleterious effect on Northern blot analysis. A lithidium chloride treatment can be added to remove this DNA (2). For RNA specific amplification using RT-PCR on the prepared RNA, we use a DNase I pretreatment of sample