Van Keulen G., Siebring J., Rembacz K.P., Hoogeveen M., Tomczynska M. and Dijkhuizen L.
Notes: These authors describe a new, quick method of isolating RNA from Streptomycetes coelicolor using five-day-old standing liquid cultures. They report higher yield, better quality RNA and increased purity compared to other methods. S. coelicolor spores were pregerminated, inoculated in liquid media and incubated at 30°C for five days. After incubation, the biomass from six 10 × 10 cm dishes was harvested by scraping the bottom of the dishes and filtering onto a membrane. Then the Mycelium was scraped from the membrane, transferred to a 1.5 ml vial, frozen in liquid nitrogen, and crushed by mortar and pestle. The resulting powder was then transferred to a tube containing 1ml Trizol, processed to extract RNA and the aqueous phase transferred to a new tube for DNase I treatment. Then 375µl of SV RNA dilution buffer was added to the nucleic acid solution, mixed and centrifuged. The resulting supernatant was mixed with 250µl cold 96% ethanol, loaded on an SV column and centrifuged for one minute. The column was then processed as described in the SV Total RNA Isolation System protocol. RNA was eluted from the column by adding 50µl of nuclease-free water and the concentration determined by spectrophotometry. To check the RNA quality, 5µg of the isolated RNA was loaded on a denaturing formaldehyde agarose gel and subjected to electrophoresis for 1 hour or used for Northern hybridizations with a nylon membrane. The 16S DNA probe was labeled with 32P-dCTP using the Prime-a-Gene® Labeling System. (3070)