We receive many calls from scientists isolating DNA from fecal samples, so I thought I’d share some tips on what we’re currently recommending for stool.
Stool is a rich microbial habitat that has a unique community to each individual. The variation in stool samples is caused by many factors. Diet can play a major role in composition and texture. Stool taken from a person with a vegetarian diet may have a higher level of polysaccharides and fiber from plant material, and therefore more inhibitors. Also, the use of antibiotics will disrupt the bacteria living in the gut and cause changes in the sample content and consistency. And of course, some foods, especially those eaten raw, for example, sushi, can impact the gut microbiome.
Just like soil, stool samples are packed with microbes that can vary depending on where the sample was collected, are rich in inhibitors, and can have different textures which will impact extraction. And most likely, if you are working with top soil, there will be some fecal material in your sample generated by earth worms, bird droppings, and other small animals. This is the reason the MO BIO kits for soil are perfectly adapted for stool.
MO BIO has several options for working with fecal material depending on the level of complexity of the sample type. The UltraClean Fecal DNA Kit is good for samples that contain a low level of inhibitors. For maximal removal of PCR inhibitors, the PowerSoil Kit is always our first choice. It is also the method used for processing samples in the Human Microbiome Project but with some changes to the protocol.
Protocol:
The SOPs for the Human Microbiome Project are listed on this webpage. The nucleic acid purification protocols are at the very bottom under the section “Metagenomic WGS”.
Changes to the PowerSoil DNA Isolation Kit are described on page 7-24 of the Manual version 11 (page 65 of the PDF).
For this protocol change, the HMP researchers are collecting 2 ml of specimen into a 50 ml Falcon tube and adding 5 ml of Bead Solution (cat# 12988-10-BS) and then distributing the sample into several samples.
We recommend, for people who want to process just a single sample, to put the stool sample directly into the PowerBead tube that comes in a PowerSoil Kit and vortex for 30-40 seconds to disperse the samples.
Next, a heating step is added in to enhance microorganism lysis:
Heat at 65ºC for 10 minutes, then at 95ºC for 10 minutes. Samples may be stored frozen at -80ºC prior to DNA extraction.
Following this treatment, the standard protocol for the PowerSoil DNA Isolation Kit is followed with a slight change at step 12 to centrifuge after Solution C3 for 2 minutes instead of one minute.
DNA is eluted in 100 ul and used for next generation sequencing.
For stool samples with the consistency closer to clay, another option that may work better is the PowerLyzer PowerSoil DNA Isolation Kit. We will be publishing research shortly that demonstrates the improved yields of DNA from clay soil using glass bead tubes. I’ll come back and post the link when it is ready.
How it Works:
One theory as to why the PowerSoil and UltraClean Fecal Kits work so well for microbial DNA isolation from fecal samples, (besides the IRT steps for removing inhibitors), may be that the final eluted DNA is enriched for microbial DNA, making PCR more sensitive. The heating steps used above will lyse the human cells first, releasing the free DNA into the sample during the bead beating steps. Microbes need to be bead beaten to break open. The free human DNA will be sheared significantly during bead beating while the microbial DNA stays high molecular weight.
For binding DNA in these kits, the binding solution (C4) captures the high molecular weight DNA only and releases RNA and small broken DNA. The broken pieces of human DNA should wash out, leaving a sample that contains a higher proportion of microbial DNA to human DNA. This means more microbial DNA goes into every reaction. For next generation sequencing, you’ll get more information from each sample from the microbiome only.
What about RNA?
For RNA from stool, the RNA PowerSoil Total RNA Isolation Kit has been referenced (see below). This method allows for a higher starting volume so that more total RNA may be recovered from each prep. If you are looking for a method to isolate RNA from small amounts of stool sample, contact us (technical@mobio.com) and we can discuss the options and provide a free sample for evaluation.
Human Microbiome references:
Dominguez-Bello, M.G., E.K. Costello, M. Contreras, M. Magris, G. Hidalgo, N. Fierer, R. Knight. In Press. Delivery mode shapes the acquisition and structure of the founder microbiota across multiple body habitats in newborns. Proc. Natl. Acad. Sci. USA. Jun 2010; 10.1073/pnas.1002601107.
Fierer, N., C.L. Lauber, N. Zhou, D. McDonald, E.K. Costello, R. Knight. 2010. Forensic identification using skin bacterial communities. Proc. Natl. Acad. Sci. USA. 107: 6477-6481.
Costello, E.K., C.L. Lauber, M. Hamady, N. Fierer, J.I. Gordon, R. Knight. 2009. Bacterial variation in human body habitats across space and time. Science. 326: 1694-1697
RNA PowerSoil Kit and stool
Exfoliated Cells in Stool: A Source for Reverse Transcription-PCR–Based Analysis of Biomarkers of Gastrointestinal Cancer
Ying Jie Yu, Adhip P.N. Majumdar, Jordan M. Nechvatal, Jeffrey L. Ram, Marc D. Basson, Lance K. Heilbrun, and Ikuko Kato. Cancer Epidemiol. Biomarkers Prev.,Feb 2008; 17: 455 – 458.
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