Total RNA was isolated from RNAprotect stabilized E. coli cultures using the RNeasy Protect Bacteria Kit (RNeasy Protect Bacteria) or from unstabilized cultures using a commercial precipitation method (Supplier EIII). To distinguish between gene expression under defined culture conditions and effects of artifactual gene induction during harvesting and RNA isolation, the RNA polymerase inhibitor rifampicin was added to half of the culture prior to RNA isolation. Differences in transcript levels with and without rifampicin therefore generally reflect the degree of RNA degradation. [A]GeneChip analysis of E.coli microarrays was carried out according to standard Affymetrix protocols. [B] The percentage of genes expressed was estimated as the number of different transcripts determined present by GeneChip analysis divided by the total number of transcripts represented on the microarray. (Data from a collaborative study with Affymetrix.)
In order to monitor mRNA degradation only, transcription was stopped by adding the RNA polymerase inhibitor rifampicin to a growing culture of E. coli. The culture was split into halves, and RNAprotect Bacteria Reagent was added to one half. Samples were left at room temperature for 0, 4, 8, and 16 minutes before centrifugation and RNA isolation. The resulting RNA was analyzed by agarose gel electrophoresis (top panel). The expression of two marker genes with different half-lives was examined by northern blot analysis. Middle panel: ompA (half life of 15 minutes); bottom panel: α-lactamase (half life of 2–5 minutes).
The RNeasy Mini spin column contained in the RNeasy Protect Bacteria Mini Kit.