Evaluation of isolation methods and RNA integrity for bacterial RNA quantitation.
RNA integrity is critical for successful RNA quantitation for mammalian tissues, but the level of integrity required differs among tissues. The level of integrity required for quantitation has not been determined for bacterial RNA. Three RNA isolation methods were evaluated for their ability to produce high quality RNA from Dickeya dadantii, a bacterium refractory to RNA isolation. Bacterial lysis with Trizol using standard protocols consistently gave low RNA yields with this organism. Higher yields due to improved bacterial cells lysis was achieved with an added hot SDS incubation step, but RNA quality was low as determined by the RNA Integrity Number (RIN). Contaminating DNA remained a problem with the hot SDS-Trizol method; RNA samples required repeated, rigorous DNase treatments to reduce DNA contamination to levels sufficient for successful real-time qRT-PCR. A hot SDS-hot phenol RNA method gave the highest RNA quality and required only two DNase treatments to remove DNA. The assessment of RNA integrity using the Agilent 2100 BioAnalyzer was critical for obtaining meaningful gene expression data. RIN values below 7.0 resulted in high variation and loss of statistical significance when gene expression was analyzed by real-time qRT-PCR. We found that RNA preparations of different quality yielded drastic differences in relative gene expression ratios and led to major errors in the quantification of transcript levels. This work provides guidelines for RNA isolation and quality assessment that will be valuable for gene expression studies in a wide range of bacteria.
Jahn
Courtney E
CE
Department of Plant Pathology, 1630 Linden drive, University of Wisconsin, Madison, Wisconsin 53706, USA.
Charkowski
Amy O
AO
Willis
David K
DK
D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D016974:Dickeya chrysanthemi / Q000235:genetics*; D020869:Gene Expression Profiling; D006358:Hot Temperature; D019800:Phenol; D016133:Polymerase Chain Reaction / Q000379:methods; D020871:RNA Stability; D012329:RNA, Bacterial / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012967:Sodium Dodecyl Sulfate
D018076:DNA, Complementary; D012329:RNA, Bacterial; D011933:Reagent Kits, Diagnostic; D019800:Phenol; D012967:Sodium Dodecyl Sulfate