RNA extraction is the most critical step in quantitative RT-PCR analysis, building cDNA libraries, or gene expression profiling. This initial step impacts every downstream application and being able to perform this task quickly and efficiently is important to lab productivity. RNA extraction from bacterial cells presents some inherent obstacles such as the Gram-positive cell wall. While Gram-negative cell walls are susceptible to most types of physical and chemical disruption used in nucleic acid extraction protocols, Gram-positive cell walls are more difficult to lyse. We evaluated Ambions’ RiboPure™-Bacteria Kit using two Gram-positive organisms,
Staphylococcus aureus and
Bacillus anthracis. B. anthracis is also capable of forming a spore that is highly resistant to most heat and chemical treatments. After using several RNA extraction methods; the RiboPure™-Bacteria Kit gave the highest yield and most consistent results but, there are some tradeoffs to using this procedure.
The RiboPure™-Bacteria Kit appears to be a streamlined and less cumbersome version of a phenol-extraction method, with several innovative steps designed to enhance RNA yield. Zirconium-oxide beads are used to disrupt cell walls by beating in a vortex mixer. A Vortex Adapter (Cat#10024) that fits on several commonly used vortex mixers is used for this purpose. I use the adaptor in conjunction with a Vortex-Genie® II. The cell lysate is then mixed with a chloroform solution (supplied in the kit) and centrifuged. The upper aqueous phase contains RNA that is easily separated, diluted with ethanol and bound to a silica filter. Wash buffers are supplied that remove contaminants and elute the RNA. An optional DNaseI treatment removes contaminating genomic DNA from the final preparation. The kit also contains RNase-free tubes for storage.
The entire procedure (including DNaseI treatment) takes around two hours. Other RNA extraction kits can take less than half the time; however, the RiboPure™-Bacteria Kit consistently gave yields ranging from 35-50ug of high quality RNA with no contaminating genomic DNA, even on difficult to work with pathogens. A buffer that allows you to store cells while stabilizing RNA, RNAlater® (Cat#7020) can be purchased separately. The use of RNAlater® seemed to have no affect on RNA recovery when processing Gram-negative microbes (Escherichia coli and Yersinia pestis) or S. aureus. However, poor RNA yields (1-5ug or less) were observed when processing B. anthracis cells with this buffer. This could be due to sporulation of B. anthracis vegetative cells. The best results were obtained when vegetative cells were processed immediately after harvesting without using RNAlater®.
One drawback to using this kit is that there is no scalability for high throughput analysis. If your goal is to get gene expression profiles from a large number of isolates or for drug discovery applications, this might not be the kit for you. I would recommend Ambions’ RiboPure™-Bacteria Kit for large amounts of high quality RNA from difficult to process pathogens such as B. anthracis. The kit is straightforward to use and troubleshooting is similar to that of typical phenol extractions.
Leo Kenefic
Research Projects Coordinator
Northern Arizona University
Biological Sciences