RNA Extraction from Microbial Cells

The extractions using the "BPC" method were performed as previously described [24 (link)], except that no 0.1 mm beads were used and the bead beating settings were not the same. All procedures were carried out at room temperature and centrifugations at 10000 g. The concentrated cell samples were centrifuged for 3 minutes and resuspended in 800 μl of water and 600 μl of buffer saturated phenol (pH 4.3). Addition of 0.5 g of 0.5 mm glass beads was followed by homogenization using a Bertin Precellys 24 (maximum speed, 20 seconds, twice). The sample tubes were then centrifuged for 10 minutes after which 750 μl of aqueous layer was transferred to a fresh tube, mixed with same volume of buffer saturated phenol (pH 4.3) and vortexed for 30 seconds. This was followed by centrifugation for 5 minutes and removal of 700 μl of aqueous supernatant that was then mixed with same volume of chloroform, in fresh tubes. After vortexing for 30 seconds, the samples were centrifuged for 5 minutes. At this point, 650 μl of aqueous layer were transferred to a fresh tube to which 65 μl of 3 M sodium acetate and 650 μl of isopropanol were added. The tubes were vortexed for 30 seconds and stored at -20°C for 10 minutes. After centrifugation for 5 minutes, the supernatant was removed and the pellet washed with 1 mL of 70% ethanol. One other centrifugation followed (5 minutes) and after removing the supernatant and air drying the RNA 50 μl of RNA storage solution (1 mM sodium citrate, pH 6.4, Ambion, Austin, USA) was added. The samples were stored at -80°C until analyzed.