QIAGEN is knee-deep in questions about DNA and RNA isolation from stool! One of the most frequently asked questions is how to store fecal samples for shipping and then storage for further processing.

 

We consulted a 2010 paper written by Dr. Rob Knight’s lab1. Dr. Knight is an expert in the microbiome field working with human gut/stool samples. In this publication, they looked at community composition and fluctuation of microbes in stool stored at various temperatures. The results from this work showed that the microbial community maintains its structure quite well, in fact, up to two weeks at room temperature with little fluctuation. Shipping of samples at room temperature or with standard cold packs should be just fine.

 

And 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 best way to keep them static. Adding preservatives with high concentrations of salts denatures proteins but also kills some microbes upon impact (unless perhaps if your diet consists of fast food and frozen pizzas on a regular basis, then your microbiome might enjoy the added salt). 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 it once had and this may contribute to change over time in storage. Any preservative for DNA and RNA 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. So keeping samples from repetitive freezing would prevent loss of the more labile species. For RNA work, freeze/thaw cycles will be especially harmful.

 

Speaking of RNA...

What about RNA isolation from stool.... a very tricky undertaking! There are a few problems with RNA isolation from fecal samples that we've identified in our lab. First, the stool is filled with dead cells, both host and microbial. The amount of degraded RNA present is very high and the recovery of this RNA will cause a lot of background on agarose gels or on the bioanalyzer. 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'll want to stick with 100% ethanol at this step.

 

Another factor can be 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 who developed a technique for removing undigested food from the stool prior to RNA isolation and this allowed for much higher yields of intact RNA to be recovered. If this protocol is of interest, let us know.

And last, stabilization as soon as possible during isolation is key to obtaining high-quality RNA from stool. To do this, there are two possible stabilizers that can be added to the lysis buffer. One is the always popular Beta-Mercaptoethanol (BME). Beta Mercaptoethanol is a strong reducing agent and RNases have disulfide bonds. Reducing agents disrupt the structure of the enzyme and render it inactive. BME can permanently destroy RNases.  Unfortunately, it does have an odor that takes some getting used to, but at least you won't need to worry anyone interrupting you in the middle of an experiment!

 

The other option is to add phenol:chloroform:isoamyl pH 7-8 (PCI) to the extraction. Only 100 ul is necessary to provide stabilization and nuclease inactivation. When added to the bead tube along with the lysis buffer prior to loading samples, the sample can be quickly protected from further degradation, especially if the samples just came out of the freezer. And if you are trying to load an entire 96 well block with stool samples, then protection as soon as possible is key, since that process can take upwards of an hour to finish.

 

For those who already have stool samples stored in RNALater, we have tested it for RNA and DNA isolation and it does work, chemistry wise, but use 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, it coats the silica membrane and results in poor recoveries.

 

That's where things stand so far in our understanding of working with stool samples, thanks to our many collaborators who worked with us on these projects. As we learn more about this unique and important sample type, more technologies in stool research as sure to develop. If you have any comments or questions, they are always welcome!

 

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:

 

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

 

1. Lauber, C., Zhou, N., Gordon, J., Knight, R., & Fierer, N. (2010). Effect of storage conditions on the assessment of bacterial community structure in soil and human-associated samples. FEMS Microbiology Letters, 307(1), 80-86. http://dx.doi.org/10.1111/j.1574-6968.2010.01965.x