Further information:
RNeasy Stabilization and Total RNA Isolation System

RNeasy Protect Bacteria Mini and Midi Kits

For RNAprotect stabilization and RNeasy purification of RNA from bacteria

　

	RNeasy Protect Bacteria Mini Kit	RNeasy Protect Bacteria Midi Kit
Format:	Mini columns with 1.5 ml and 2 ml collection tubes	Midi columns with 15 ml collection tubes
Sample size*
Bacteria:	<7.5 x 108	5 x 108 – 7.5 x 109
Volume of RNAProtect Bacteria Reagent:†	50 ml (50-prep kit)	20 ml (10-prep kit)
Binding capacity:	Up to 100 µg RNA	Up to 1 mg RNA
Yield*
E. coli:	100 µg (6 x 108 cells)	160 µg (1 x 109 cells)
B. subtilis	15 µg (1 x 108 cells)	75 µg (5 x 108 cells)
Elution volume:	30–100 µl	300–500 µl

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 figure "RNAprotect Bacteria Reagent Prevents mRNA Degradation"). 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.

RNeasy Kits provide the highest-quality RNA with minimum copurification of DNA. For certain RNA applications that are sensitive to very small amounts of DNA, the residual amounts of DNA remaining can be removed using the RNase-Free DNase Set for convenient on-column DNase treatment during the RNeasy procedure.

* Amounts can vary due to species, growth conditions used, etc. Since the RNeasy procedure enriches for RNA species >200 nt, RNA yield does not include 5S rRNA, tRNAs, or other low-molecular-weight RNAs.
† Stabilization requires 2 volumes RNAprotect Bacteria Reagent per 1 volume bacterial culture.

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: beta lactamase (half life of 2–5 minutes).

操作手册:RNAprotect Bacteria Reagent Handbook (PDF version, 236 KB)

         RNeasy Mini Handbook 6/01 (PDF version, 351 KB)

         RNeasy Midi/Maxi Handbook 06/01 (PDF version, 345 KB)

Ordering Information Product Contents Cat. No. RNeasy Protect Bacteria Mini Kit (50) RNeasy Mini Kit (50) and RNAprotect Bacteria Reagent (2 x 100 ml) 74524 RNeasy Protect Bacteria Midi Kit (10) RNeasy Midi Kit (10) and RNAprotect Bacteria Reagent (2 x 100 ml) 75552 RNAprotect Bacteria Reagent RNAprotect Bacteria Reagent (2 x 100 ml) 76506