Efficient isolation of total RNA from antibiotic-producing bacterium Amycolatopsis mediterranei.
RNA extraction from antibiotic-producing actinomycetes can be a difficult and time-consuming process due to their special peptidoglycans cell wall composition and the short life of RNA. Hence, the rapidity of cellular lysis and complete inhibition of RNase are of particular importance for isolating intact RNA of high quality. The genus of Amycolatopsis mediterranei produces many clinically important antibiotics, such as rifamycin and vancomycin; however, the available methods for bacterial RNA isolation did not work very well with this genus. In this report, we described a new method for RNA isolation using the combination of LiCl, urea and guanidinium thiocyanate to disrupt the cell wall of Amycolatopsis. Compared with earlier published RNA isolation methods, the method gave higher yields of pure and intact RNA. About 1 microg total RNA free of DNA contamination can be obtained from 1 mg wet weight of A. mediterranei. The integrity of the RNA was demonstrated by formaldehyde agarose gel electrophoresis and Northern blot analyses.
Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, 200032, Shanghai, PR China.
Yufeng
Y
Yao
Weiwen
W
Zhang
Ruishen
R
Jiao
Guoping
G
Zhao
Weihong
W
Jiang
D000192:Actinomycetales / Q000235:genetics*; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001431:Bacteriological Techniques; D015152:Blotting, Northern; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000032:analysis / Q000302:isolation & purification*
D000900:Anti-Bacterial Agents; D012329:RNA, Bacterial