1Department of Pediatrics, University of Texas Southwestern Medical Center, 2Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 3Department of Pediatrics and Microbiology, University of Texas Southwestern Medical Center
Lopez-Medina, E., Neubauer, M. M., Pier, G. B., Koh, A. Y. RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract. J. Vis. Exp. (55), e3293, doi:10.3791/3293 (2011).
Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host’s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.
1. Murine Model of P. aeruginosa GI Colonization and Dissemination
2. Harvesting Murine Cecal Luminal Contents
3. Bacterial RNA Isolation
4. DNase Treatment (Turbo DNA-Free, Applied Biosystems)
5. RNA Cleanup Step (Qiagen, RNeasy Kit)
6. Representative Results
The amount of bacterial total RNA recovered by using this protocol is approximately 2-3 μg from two cecums. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. RNA purity is routinely assessed by measuring the 260nm/280nm ratio7 of the sample, yet this method provides no information about RNA integrity. The Agilent Bioanalyzer is a microfluidics-based platform for sizing, quantification and quality control of DNA, RNA, proteins and cells; and utilizes an RNA integrity metric known as RNA Integrity Number (RIN)8. RNA extracted with this protocol produces 260/280 ratios ranging from 1.7 to 2.0 and RIN values ≥ 7.0. An example of an Agilent Bioanalyzer analysis of the bacterial RNA recovered by this protocol is shown in Figure 1.
Figure 1. Agilent Bioanalyzer electropherogram and gel-like image of Pseudomonas aeruginosa total RNA sample isolated and recovered from murine cecal contents. RIN 8.0.
The RNA extraction method described here allows for recovery of sufficient quantities of high-quality Pseudomonas aeruginosa total RNA harvested from the murine GI tract. This method is not restricted to P. aeruginosa and can potentially be applied to other bacteria. The recovery of sufficient microbial organisms from the intestine will vary significantly from organism to organism. In our murine model, P. aeruginosa typically colonizes the murine GI tract at levels between 5 x 107 to 5 x 108 cfu/g feces4. Since the recovered cecal contents are roughly 0.5 gram, the estimated number of P. aeruginosa recovered from two cecums is between 5 x 107 to 5 x 108 cfu. If other microorganisms are used, it would be prudent to verify GI colonization levels and then calculate the number of cecums needed to recover the targeted number of cfu. It is also important to note that when using this particular murine model, antibiotic-treated mice not infected with PA have no quantifiable amounts of RNA isolated from their cecal contents.
Our murine model of Pseudomonas aeruginosa gastrointestinal colonization and dissemination attempts to emulate the pathogenesis of P. aeruginosa bacteremia in cancer and stem cell transplant patients. In this patient population, commensal flora is often depleted secondary to antibiotic or chemotherapeutic treatment (e.g the antibiotic depletion of GI commensal flora) resulting in overgrowth of pathogenic microbes (e.g. mono-association with P. aeruginosa) and then subsequent dissemination after immune suppression. The advantages and limitations of this murine model have already been addressed previously4. The purpose of this current study is to provide a methodology for isolating microbial total RNA from the GI tract. This protocol can easily be adapted to other murine models that study other aspects of microbial pathogenesis in the GI tract (i.e. commensal flora interactions, bacterial effects on inflammatory bowel disease, etc.).
One advantage of this method is the incorporation of multiple lysis steps including freeze/thaw, mechanical disruption (pulverizing with mortar/pestle, homogenization), boiling, and chemical lysis (e.g. SDS). Despite the multitude of lysis steps, some microorganisms (notably Gram-positive bacteria and fungi/yeast) may require additional mechanical disruption. After the hot lysis/acid phenol-chloroform incubation (Step 3.5), the addition of beads (0.1 mm for bacteria and/or 0.5-0.7 mm for yeast) and a subsequent bead-beating step should be sufficient to lyse these organisms9, 10.
Given the complex nature of materials recovered from the murine cecum, the repeated cold acid-phenol/chloroform extractions (Step 3.7 to 3.9) are absolutely required in order to achieve acceptable RNA quality and integrity for further downstream reactions. Between 3-5 cold acid-phenol/chloroform extractions may be required before the white interface between the aqueous and organic phases is eliminated. Finally, the combination of both the DNase treatment (Step 4) and RNeasy Cleanup Protocol (Step 5) are essential for removing contaminating DNA and small non-mRNA (5s and tRNA). As stated previously, the RNA recovered by utilizing this protocol is of sufficient quantity and quality for subsequent transcription profiling 3, qPCR (unpublished), and RNA Seq (unpublished) experiments.
No conflicts of interest declared.
This work was funded by the National Institutes of Health grants AI62983 (AYK), AI22535 (GPB).
|Mortar and Pestle||Fisher Scientific||12-947-1|
|Oak Ridge centrifuge tubes (50 ml)||Nalge Nunc international||3119-0050|
|Diethyl pyrocarbonate (DEPC)||Sigma-Aldrich||40718|
|3M Sodium Acetate||Ambion||AM9740|