Further information:
RNAlater
and RNAprotect Systems
For stabilization of gene expression profiles in bacteria
When using traditional methods for cell harvesting and RNA isolation, two major effects can lead to vast changes in bacterial expression profiles, causing artifacts in gene expression analyses. Firstly, enzymatic degradation of RNA leads to reduction or loss of many transcripts. The reduction is particularly significant in bacterial mRNA molecules because they usually have very short half lives of only a few minutes. Secondly, genes can be induced during handling and processing of samples, leading to higher expression levels of certain genes. Use of RNAprotect Bacteria Reagent overcomes these problems by providing immediate stabilization prior to RNA isolation (see figures). It is suitable for use with a wide range of bacterial species, both Gram positive (e.g., Staphylococcus aureus and Mycobacterium avium) and Gram negative (e.g., Escherichia coli and Salmonella typhimurium). Bacteria grown in either minimal or complex medium can be used.
Two volumes of reagent are added directly to 1 volume of bacterial culture prior to RNA isolation, providing immediate stabilization of RNA. The stabilization allows time for efficient bacterial lysis using a choice of protocols: enzymatic lysis, mechanical disruption, or a combination of both methods. QIAGEN recommends the Mixer Mill MM 300 for efficient mechanical disruption.
GeneChip Analysis Shows Accurate Gene Expression Profiles with RNAprotect Stabilization
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.)
RNAprotect Bacteria Reagent Prevents mRNA Degradation
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 two 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). 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: ¦Á-lactamase (half life of 2¨C5 minutes).
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Bacteria Reagent Handbook (PDF version, 236 KB)
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