Integrity Analysis of RNA Isolated From RNAlater Stabilized Bacteria
To demonstrate the effectiveness of RNAlater at preserving RNA in intact bacteria, samples from five bacterial species, Escherichia coli, Pseudomonas aeruginosa, Campylobacter fetus, Bacillus subtilis and Staphylococcus aureus, were suspended in RNAlater and incubated at 25C or 4C for up to one week. RNA was isolated from the bacteria, and RNA integrity was evaluated by analysis on the Agilent 2100 bioanalyzer and by filter based array analysis.
Log phase Bacillus subtilis cells were harvested by centrifugation, resuspended in RNAlater (or TE as a negative control), and incubated for four hours at room temperature or one week at 4C. RNA was isolated from cells by bead-beating with zirconium/silica beads in RNAwiz (Ambion). RNA integrity was evaluated using the RNA 6000 LabChip and the Agilent 2100 bioanalyzer (Figure 1, A-D). Evaluation of the electropherogram s obtained from the RNAlater preserved samples, Figure 1C and 1D, clearly demonstrate that the quality of RNA recovered from B. subtilis cells stored in RNAlater is indistinguishable from the positive control sample (Figure 1A). The RNA from B. subtilis stored in TE buffer for four hours is severely degraded (Figure 1B) .
Figure 1. Bacterial RNA Stability. (A) Total RNA isolated from B. subtilis and processed immediately. The ratio of 23S:16S rRNA is 1.8. No RNA degradation is apparent. (B) Total RNA isolated from B. subtilis incubated in TE for 4 hr at 25C. Extensive degradation of 16S and 23S rRNAs is apparent. (C) Total RNA isolated from B. subtilis incubated in RNAlater for 4 hr at 25C. The ratio of 23S:16S rRNA is still 1.8. No RNA degradation is apparent. (D) Total RNA isolated from B. subtilis incubated in RNAlater for 1 week at 4C. The ratio of 23S:16S is still 1.8. No RNA degradation is apparent.
RNAlater is Effective at Preserving Bacterial RNA for Gene Expression Profiling With Arrays
To determine if storage of bacteria in RNAlater affects gene expression patterns, we compared the expression profile obtained with RNA isolated from B. subtilis that had been stored overnight in RNAlater at 25C (Figure 2A) with the profile from RNA prepared from freshly harvested B. subtilis (Figure 2B). A comparison of these replicate arrays demonstrates that RNAlater is extremely effective at preserving gene expression profiles in bacteria. Similar results were obtained when arrays were probed with RNA prepared from B. subtilis cells that were stored in RNAlater for one week at 4C. RNAlater is thus an ideal solution for maintaining the expression profile of bacterial mRNA when immediate RNA isolation is not possible or practical.
Figure 2. Storing Bacteria in RNAlater Does Not Affect RNA Expression Profiles. cDNA used for hybridization to the array on the right (B) was prepared from total RNA (5 g) isolated from freshly harvested B. subtilis. cDNA used for hybridization to the array on the left (A) was prepared from total RNA (5 g) isolated from B. subtilis cells incubated in RNAlater overnight at 25C.
Ambion recommends the following protocol for preserving bacterial RNA using RNAlater. Cells should be collected by centrifugation and resuspended in RNAlater immediately after harvesting. Resuspend cell pellets from 2 ml of logarithmic phase cell culture in 0.5 ml - 1 ml of RNAlater. RNAlater-treated cells can be stored up to 8 hrs at 25C or up to a week at 4C. For longer storage, cells should be pelleted after incubation in RNAlater for one hour, flash frozen, and stored at 80C.