Exponentially growing culture held in M9 minimal salts for 120 min. then allowed to recover in LB for 90 min., after which total RNA was isolated.
Extracted molecule
total RNA
Extraction protocol
Epicentre MasterPure RNA Purification kit and Affymetrix protocol for E. coli Sense arrays (mRNA enrichment protocol)
Label
PEO-iodeacetyl-Biotin
Label protocol
Affymetrix E. coli Sense arrays
Hybridization protocol
Affymetrix
Scan protocol
Affymetrix
Description
Overnight culture was diluted 1000-fold with fresh LB medium and cultured further. Log phase culture was diluted to a cell density of 2 X 108 cells/ml in M9 salts and incubated at 37°C for two hours, after which it was resuspended in LB broth for 90 minutes. This treatment served as a mock treatment for cultures incubated with a chemical in a parallel study. Total RNA was isolated from cells by extraction using the MasterPure RNA Purification Kit (Epicentre Technologies) followed by DNAse digestion according to the manufacturer’s protocol. The isolated total RNA was quantitated by absorption at 260 nm (typical yield from a 15 ml culture was 250-500 µg of total RNA), and the purity was determined by the ratio of absorption values at 260/280nm. RNA quality was determined by formaldehyde agarose gel electrophoresis (1.2% agarose in FA Buffer pH 7.0 (20 mM 3-[N-morpholino]propanesulfonic acid, 5 mM sodium acetate, 1 mM ethylenadiaminetetraacetic acide (EDTA))) or by analysis on an Agilent 2100 Bioanalyzer. All samples visualized by gel electrophoresis or by the bioanalyzer electropherogram showed clear distinct bands correlating to 16S and 23S ribosomal RNA, indicating that no detectable RNA degradation occurred and that RNA integrity was maintained throughout the RNA isolation procedure. mRNA was enriched from total RNA as described in the Affymetrix GeneChip Expression Analysis Technical Manual for GeneChip E. coli Sense Genome Arrays.
