The value of PCR to forensics has been known for a long time; but now, getting purified DNA and RNA samples from soil and fecal samples is becoming more important and commonplace as tests for environmental impacts, disease spread, and even biomarkers for colon cancer become more prevalent. And if you thought getting nucleic acids out of relatively clean samples, like lab grown organisms, then you ain’t seen nothing yet. Soil, feces, blood and other tissues create some unique obstacles to extracting PCR-quality DNA and RNA. Yield is often an issue, and that’s due to the biggest problem with these types of samples: PCR inhibition.
All of these samples contain inhibitors, that is molecules, salts or other chemicals that directly interfere with DNA itself or with DNA polymerases. Here are the culprit inhibitors for each sample type:
Fecal samples: bile salts and polysaccharides
Soil and plant samples: humic acid and polysaccharides
Blood: heme, hemoglobin, IgG and lactoferrin.
All samples (inhibitors introduced during the purification process): KCl, NaCl, sodium deoxycholate, SDS, and ethanol.
The special case of RNA
RNA is particularly difficult to isolate from feces and other such samples. Since feces contain a lot of RNase-rich dead cells, a lot of RNA is degraded and needs to be removed. Also, undigested food in feces reduces the relative amount of microbial mass to work with, and also needs to be removed. And of course, the RNases need to be removed before you can work with the isolated RNA.
Cold storage? No!
Rob Knight, a scientist working with the American Gut Project, wrote in 2010 that freezing fecal samples is unnecessary, and may affect the microbial balance of your sample. Microbes in these samples seem to stay stable at room temperature, and shipping the samples at ambient temperature or perhaps with a cold ice pack (not dry ice) will maintain the integrity of the sample. This also will maintain the normal microbial environment in the sample, which could be crucial to microbiome research.
A stone’s throw away
Where can you find the materials for effective DNA/RNA purification from feces and soil (and others)? You could start by throwing a rock. Depending on where you are, you might hit about 10 commercial PCR purification kits that cater to these challenging samples. About three years ago, a group from the U.S. Centers for Disease Control tested three DNA purification kits—the MoBio UltraClean soil DNA isolation kit, the Qiagen QIAamp DNA minikit, and the Qiagen QIAamp DNA stool minikit. Still another kit is the Promega DNA IQ System.
The CDC found that the MoBio kit removed inhibitors from 35% of their samples, while the QIagen tissue protocol removed inhibitors from just 4% of its samples. The Qiagen stool kit, however, removed inhibitors from 96% of its samples. Using the kits together resulted in total inhibitor removal, however. In fact, Promega advises using multiplex real-time PCR to quantitate DNA, which allows for using a positive control to detect any inhibitors.
Fitzpatrick, K.A., Kersh, G.J., and Massung, R.F. (2010). Practical method for extraction of PCR-quality DNA from environmental soil samples. Applied and Environmental Microbiology, 76(13), 4571-4573.
Lauber, C.L., Zhou, N., Gordon, J.I., 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(1), 80-86.
Bessetti, J. (2007). An introduction to PCR inhibitors. Profiles in DNA. www.promega.com.