A rapid novel method for the extraction of RNA from wild-type and genetically modified kanamycin resistant mycobacteria.
RNA extraction from mycobacteria can be a difficult and time consuming process due to their unique mycolic acid cell wall composition and the short half life of RNA. Rapid cell wall disruption is essential to isolate intact high quality RNA. Genetically modified Mycobacteria smegmatis resistant to kanamycin demonstrate an altered cell wall composition which impairs the quality of RNA obtained from these mycobacteria. In this report we describe a method of RNA extraction using the detergent Catrimox-14 resulting in high yields of pure, undegraded RNA in less than 1 h. Yields of 25-30 microg RNA per 1x10(9) cells were consistently obtained from both wild-type and genetically modified M. smegmatis. The integrity of this RNA is demonstrated by gel electrophoresis, Northern blot and cDNA analysis.
Payton
M
M
University Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, UK.
Pinter
K
K
D015152:Blotting, Northern; D004251:DNA Transposable Elements; D018076:DNA, Complementary / Q000032:analysis; D003902:Detergents; D005818:Genetic Engineering; D007613:Kanamycin Resistance / Q000235:genetics*; D008967:Molecular Biology / Q000379:methods; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000235:genetics*; D000644:Quaternary Ammonium Compounds; D012329:RNA, Bacterial / Q000032:analysis / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050337:Trimethyl Ammonium Compounds
D004251:DNA Transposable Elements; D018076:DNA, Complementary; D003902:Detergents; D000644:Quaternary Ammonium Compounds; D012329:RNA, Bacterial; D050337:Trimethyl Ammonium Compounds; C009410:tetradecyltrimethylammonium