QIAGEN receives so many questions about DNA and RNA isolation from stool! One of the most frequently asked questions is how to store fecal samples for shipping and for further processing. Since storage conditions are a key component of DNA-based microbial community analysis methods, we will share the best methods with you. We consulted a 2010 paper written by Dr. Rob Knight’s lab (1). Dr. Knight is an expert in the microbiome field working with human gut/stool samples. In this publication, the researchers determined whether differences in short-term sample storage conditions affect the assessment of bacterial community composition and diversity in stool stored at various temperatures. This paper demonstrated that the phylogenetic structure and diversity of communities in individual samples was not significantly influenced by storage temperature or duration of storage. There was minimal fluctuation in the relative abundances of most taxa after storage for up to 14 days at room temperature. These results suggest that many samples collected and stored at room temperature or with standard cold packs may be useful for microbial community analyses.
This makes sense from an ecology standpoint. The stool matrix is a complex environment with specific ratios of components such as bile, bilirubin, heme and digested food unique to each individual. Maintaining the natural environment of the microbes would be the most efficient way to keep them static. Adding preservatives with high concentrations of salts denatures proteins but also kills some microbes upon impact. The addition of denaturing chemicals will change the environment drastically, such that the microbes that do survive the preservative no longer have the same ecosystem they once had and this may contribute to change over time in storage. Any preservative used for DNA and RNA isolation work would need to keep the cells from breaking while maintaining the diversity of microbial life in the stool sample.
Freeze/thaw cycles of the sample are also detrimental to microbes since the formation of ice crystals can cause cells to break during thawing. Keeping samples from repetitive freezing would prevent loss of the more labile species. For RNA work, freeze/thaw cycles are especially harmful. In general, RNA isolation from stool can be a very tricky undertaking. We have identified several problem areas with RNA isolation from fecal samples in our R&D lab:
• Large quantities of degraded RNA
• Undigested food and reduced RNA yield and quality
• Stabilization of RNA and RNA quality
The first issue is that stool contains many dead cells, both host and microbial. The amount of degraded RNA present is very high and the recovery of this RNA can cause a lot of background on agarose gels. This low molecular weight RNA can be removed from spin filter purification preps by switching to 70% ethanol instead of 100% ethanol during binding. However, if you are interested in microRNAs, you should continue to use the 100% ethanol at this step.
The second issue is that the amount and type of undigested food in the stool sample. This adds bulk and weight thus reducing the amount of actual microbial mass going into the prep. We have a customer-developed technique for removing undigested food from the stool prior to RNA isolation that allowed for much higher yields of intact RNA to be recovered. If this protocol is of interest, let us know below in the comment section.
The third issue concerns stabilization. Stabilization as soon as possible during isolation is critical to obtaining high-quality RNA from stool. There are two possible stabilizers that can be added to the lysis buffer to properly stabilize the sample. One is beta-mercaptoethanol (BME), a strong reducing agent. Reducing agents disrupt the structure of enzymes and render them inactive. BME can permanently destroy RNases since they have disulfide bonds. The other option is to add phenol:chloroform:isoamyl, pH 7–8 (PCI), to the extraction. Only 100 µl is necessary to provide stabilization and nuclease inactivation. When added to a bead tube along with the lysis buffer prior to loading samples, the sample can be quickly protected from further degradation, especially if the samples recently came out of the freezer. If you are trying to load an entire 96 well block with stool samples, then protection as soon as possible is key, since the process can take upwards of an hour to complete.
What about RNAlater? Storing stool samples in RNAlater is fine, we have tested it for RNA and DNA isolation; however, we recommend using only 100 mg of sample in this case. The salt in RNAlater interacts with the stool matrix and causes a precipitate. If too much sample is used, RNAlater coats the silica membrane and results in poor recoveries.
Once you’ve stabilized your stool samples and are ready to isolate nucleic acids, QIAGEN offers several options for isolation of high-quality, pure nucleic acids from stool and gut samples (Table 1):
Table 1. QIAGEN kits for isolation of nucleic acids from stool or gut samples.
|QIAGEN kit||Nucleic acids||Sample type||Throughput|
|QIAamp PowerFecal DNA Kit||DNA||Stool, gut, biosolids||Manual|
|MagAttract PowerMicrobiome DNA/RNA Kit||DNA and RNA||Stool, gut, biosolids||High-throughput, hands-free|
|AllPrep PowerViral DNA/RNA Kit||DNA and RNA||Wastewater, stool||Manual|
|RNeasy PowerMicrobiome Kit||RNA (and DNA if you skip DNase step)||Stool, gut, biosolids||Manual|
Missed our microbiome webinar on simplifying and streamlining DNA extraction for samples with high inhibitor content and subsequent microbial community analyses? Download the recording now!
- 1. Lauber, C., Zhou, N., Gordon, J., Knight, R., and Fierer, N. (2010). Effect of storage conditions on the assessment of bacterial community structure in soil and human-associated samples. FEMS Microbiology Letters 307, 80–86. Link
Authors: This post was originally published on mobio.com, and has been updated and modified by Heather Martinez and Miranda Hanson-Baseler.