One of the most difficult sample types we work with in our labs is soil. When developing products and methods for isolation of microbial DNA and RNA from soil, we have to take into account the wide diversity of soils in regards to their organic content, texture, pH, and where the soil was collected. These factors and more impact the microbial load and therefore, the yields of DNA and RNA that can be obtained.
In this article, I’ll dish the dirt on some of the technologies available for isolating DNA and RNA from soil. For more information on the types of organisms found in soil, here is a short primer on soil microbiology.
A major factor that impacts DNA and RNA isolation from soil is the level of humic substances (humic acids, fulvic acids, and humins). Humics are formed by the degradation of organic matter (plant and microbial material) and results in the dark color of soil (due in part to the quinone structure of the molecule) (1). They are large stable macromolecules that can vary in size and have both phenolic and carboxylic groups. Humics will chelate multivalent cations, making them readily available for microbes and plants that need them (2). A great overview of humic substances can be found here.
Because humic and fulvic acids are large water-soluble anionic polymers like DNA and RNA, they will co-purify in nucleic acid extraction protocols (3). For this reason, the humic substances are best removed before the final purification.
Inhibitor Removal Technology (IRT):
Inhibitor removal technology is the MO BIO patented method for removal of humic substances as well as polyphenolics and polysaccharides from samples. The system works by using changes in pH to solubilize and release charged molecules followed by removal of protein and then dropping the pH to precipitate the insoluble large macromolecules. The nucleic acids do not precipitate and are cleared of inhibitors. This also works for molecules like heme in blood and fecal samples, melanin in skin, and dyes from clothing from forensic samples such as blood stained clothing.
Lysis of microorganisms:
Removal of inhibitors is a major issue but the second most important issue is achieving strong lysis of the microbial species in the soil so that there is an accurate representation of the microbial community. Mechanical lysis provides the fastest and most efficient method for lysing bacteria and fungus in soil. At MO BIO Labs, we prefer to use the Vortex Genie 2 because it offers many advantages over the high-powered bead beating instruments.
A major advantage of using the vortex is that is provides higher quality (molecular weight) DNA because it uses less force over longer time (10 minutes) to pulverize cells. The lower force means that the sample doesn't heat up excessively during lysis and reduces damage due to over-heating of DNA and RNA. The combination of the MO BIO lysis solutions for soil with the vortex allows for high yield extraction of DNA and RNA with the least amount of damage. Plus, the solutions used for lysis set up the sample to be purified with IRT. For metagenomic studies where high molecular weight DNA is desired, the vortex is the best choice.
On a budget?
For fieldwork, the vortex is small and inexpensive making it more convenient for labs on a budget or when samples are processed in field labs. The vortex is lightweight and can accommodate up to 24 samples at one time. To make high throughput processing easier, MO BIO developed a series of adapters that fit on the Vortex Genie 2. Vortex adapters are available for all sized tubes.
Need stronger lysis?
While the lysis using the vortex and our solutions will do the trick for most organisms, people working with spores or fungus sometimes want to use an even stronger method for lysing cells. For these types of samples, we typically use a heating step before the vortex. Incubating the soil in lysis buffer for 10 - 15 minutes at 65oC-70oC will do the trick (4, 5). Freeze thaw cycles prior to vortex is another way to enhance the lysis of tough spores and fungal cells.
What about high powered bead beating?
High powered bead beating is another approach sometimes used for tough organisms but will result in greater shearing of DNA and heat induced RNA degradation. In our own lab, we do not observe greater DNA yields using the bead beater instrument for DNA extraction from soil based on gel analysis. However, Nanodrop readings will often register a higher absorbance at wavelength 260 when the DNA is sheared. In addition, using plate counting for measuring the number of cultureable bacteria after using either the Precellys or the vortex for lysis of bacteria in soil, the number of viable organisms were the same. Since high powered bead beating will increase the chance of release of plant and insect DNA into the sample, thus diluting the microbial DNA content, we avoid this method for soils.
In this introductory blog, we've covered two main issues involved in soil molecular work. In the next couple of blogs, we'll go into detail on the DNA extraction methods and I'll present some tips and tricks for getting high yields and purity using the PowerSoil DNA Kit and the RNA PowerSoil Kit.
Thanks for reading. If you have any questions or would like a specific topic covered, please leave a comment.
1. A. Piccolo (2002). "The Supramolecular structure of humic substances. A novel understanding of humus chemistry and implications in soil science". Advances in Agronomy 75: 57–134.
2. F.J. Stevenson (1994). Humus Chemistry: Genesis, Composition, Reactions. John Wiley & Sons, New York
3. Techniques in Microbial Ecology, Burlage, Robert S., Ronald Atlas, David Stahl, Gill Geesey, and Gary Sayler, editors. 1998. Oxford University Press, New York, Page 277
4. Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate
Pascal E. Saikaly, Morton A. Barlaz, and Francis L. de los Reyes III
Applied and Environmental Microbiology, October 2007, p. 6557-6565, Vol. 73, No. 20
5. Evaluation of five commercial nucleic acid extraction kits for their ability to inactivate Bacillus anthracis spores and comparison of DNA yields from spores and spiked environmental samples
Leslie A. Dauphin, Benjamin D. Moser
Journal of Microbiological Methods, Volume 76, Issue 1, January 2009, Pages 30-37