Isolation of RNA from mammalian cells is usually straightforward; however; bacterial cells may pose additional problems. I have used Qiagen’s RNeasy Mini Kit extensively on mammalian cells with favorable results and I recently had the opportunity to evaluate this kit using two Gram-positive bacteria,
Staphylococcus aureus and
Bacillus anthracis. Having used many different methods to isolate RNA, I’ve found the RNeasy Kit to be among the least time-consuming and the most user-friendly. However, there are limitations that should be considered.
The RNeasy Mini Kit uses a modified guanidine isothiocyanate and silica membrane isolation procedure. The kit contains most everything you need with the exception of β-mercaptoethanol (added to the lysis buffer) and 100% ethanol (added to the wash buffers). The kit also contains RNase-free collection tubes for storage. The cell lysis step is crucial for good recovery of nucleic acids. Cell lysates for mammalian cells are generated by using a QiaShredder (catalog# 79654) and centrifugation after addition of a lysis buffer. Lysing bacterial cell walls requires lysozyme and TE Buffer, which are not supplied with the kit. The two Gram-positive bacteria required longer incubation times (~30 minutes) to lyse the cell walls than noted in the manual. Also, overloading the column with bacterial cells negatively impacted the yield of RNA regardless of incubation times in lysis buffer. Lysates are added to the top of the RNeasy column and bound to the filter by centrifugation. Once bound, the RNA is washed and eluted. The entire process takes less than 30 minutes for mammalian cells and approximately 60 minutes to process Gram-positive cells.
I used the RNeasy Kit to extract RNA from bone marrow and peripheral blood. RNA yields were typically around 20ug from 107 cells. We used this RNA in random-primed RT-PCR reactions followed by verification of amplicons by Southern-blot hybridization. I never found contaminating DNA to be an issue in this scenario. However, contaminating DNA was an issue for bacterial gene expression profiling as the low level genomic DNA interfered with hybridization to the chip. To remedy this, Qiagen offers an optional on-column DNase treatment (catalog # 79254).
A buffer that allows you to store cells while stabilizing RNA, RNAlater® (Cat# 76106) can be purchased separately or as part of the RNeasy Protect Mini Kits (Cat # 74126). The use of RNAlater® seemed to have no affect on RNA recovery when processing mammalian cells. However, poor RNA yields (1-5ug or less) were observed when processing B. anthracis with this buffer. This was probably due to sporulation of B. anthracis vegetative cells and has been observed with other RNA stabilization buffers as well. The best results were obtained when vegetative cells were processed immediately after harvesting without using RNAlater®. A typical yield from 109 vegetative cells averaged 35ug and was contingent upon the age of the culture.
This kit has an upgraded counterpart (RNeasy 96) in 96-well format 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 kit may warrant a closer look. I would recommend Qiagen’s RNeasy Kit for high throughput applications involving mammalian cells or bacteria. This kit has the most straightforward protocol and training new technicians in its use takes the least amount of time.
Leo Kenefic
Research Projects Coordinator
Northern Arizona University
Biological Sciences