RNAlater™ is the worldwide standard for the preservation of RNA in intact animal tissues, organs, and cells. RNAlater eliminates the need to immediately process tissue samples or to freeze samples in liquid nitrogen for later processing. Here we demonstrate that RNAlater provides the same outstanding benefits when used with bacteria. It immediately stabilizes RNA expression profiles in both gram-positive and gram-negative species.
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 25°C or 4°C 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.
Integrity Analysis of RNA Isolated From RNAlater™ Stabilized Bacteria
Log phase Bacillus subtilis cells were harvested by centrifugation, resuspended in RNAlater™ (or TE as a negative control), and incubated for 4 hrs at room temperature or one week at 4°C. 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 electropherograms 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 hrs is severely degraded (Figure 1B).
Figure 1A. Total RNA isolated from B. subtilis processed immediately. The ratio of 23S:16S rRNA is 1.8. No RNA degradation is apparent.
Figure 1B. Total RNA isolated from B. subtilis was incubated in TE for 4 hr at 25°C. Extensive degradation of 16S and 23S rRNAs is apparent.
Figure 1C. Total RNA isolated from B. subtilis incubated in RNAlater for 4 hr at 25°C. The ratio of 23S:16S rRNA is still 1.8. No RNA degradation is apparent.
Figure 1D. Total RNA isolated from B. subtilis incubated in RNAlater for 1 week at 4°C. 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 25°C (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 4°C. 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. Replicate Panorama™ Bacillus subtilis arrays (Sigma Genosys) were hybridized with 33P-labeled cDNA generated from 5 µg of B. subtilis total RNA. cDNA used for hybridization to the array on the right was prepared from total RNA isolated from freshly harvested B. subtilis. cDNA used for hybridization to the array on the left was prepared from total RNA isolated from B. subtilis cells incubated in RNAlater overnight at 25°C.
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 approximately 8 hrs at 25°C or up to a week at 4°C. For longer storage, cells should be pelleted after incubation in RNAlater for one hour, flash frozen, and stored at 80°C.