As part of our research on the best practices for soil microbial DNA extraction, we collect a wide variety of samples for product development. So when we were developing the protocols for the PowerLyzer 24 Homogenizer 24 Homogenizer, we wanted a protocol that worked for most of the samples tested. Our work on homogenization and bead tubes previously showed that depending on the soil, sometimes a different bead type could give you an increased yield of DNA. We decided to do a similar study using the PowerLyzer 24 Homogenizer to ask the question: what is the difference in DNA yields and integrity using high powered bead beating between two different soils using the same protocols? It is not uncommon for people to simply adopt a protocol from a paper for their soil type without doing any optimization. But, does one protocol really work best for every soil?
Today we would like to share with you some of our own results in our quest to answer these questions. We wanted to compare the results of DNA yields and integrity from two different soil types; one with high clay content and one with high carbon content using high powered bead beating (the PowerLyzer 24 Homogenizer) and comparing two different bead types; 0.1 mm glass beads (cat# 13118) vs. 0.7 mm garnet beads (cat# 13123). The results were very surprising.
Methods: We received a variety of soils from the California Polytechnic State University Soil Science Center. We chose two soils for this study that were similar in clay content but different in carbon content. One of the soils was 45% clay and low in carbon (2.5%) and the other was 40% clay but higher in carbon (5%). The carbon content results in a difference in microbial biomass. The high carbon content soils have the highest biomass and therefore higher yields of DNA.
We compared our 0.7 mm garnet bead tubes which come in the DNeasy PowerSoil Kit with the 0.1 mm glass bead tubes which come with the DNeasy PowerLyzer PowerSoil Kit. We performed DNA isolations on soils starting at a speed of 2000 RPM on the PowerLyzer 24 Homogenizer and increasing all the way to 5000 RPM, the highest setting on the PowerLyzer 24 Homogenizer, and all preps were run for 45 seconds.
DNA was isolated using the DNeasy PowerSoil Kit and the DNA quantified on a Nanodrop and run on gels to check for integrity. We plotted the yields on bar graphs with the data represented by glass beads in blue and the garnet in red.
High Clay, Low Carbon Soil
This first figure shows the results of the higher clay soil on the PowerLyzer 24 Homogenizer.
We see that the glass beads in general extract more DNA from this soil and this peaks at a speed of 3900-4200 RPM (lanes 14 and 16). What is interesting is that when you use speeds higher than 4200 RPM, yields actually go down. This demonstrates that you can beat a soil too much or too hard and lose DNA. There may be an optimal speed or time for your soil that you would not want to go past to get the best yields.
We also see that at the lower speeds, slower than 3900 RPM, the glass and garnet beads were extracting equal yields. There really was not a big difference in yields between the two. Why could this be?
One theory is that perhaps the soils have two predominat types of communities: easy to lyse and hard to lyse. It may be that the easy to lyse organisms break open with either bead type at the lower speed and then the boost at 3900 with the glass beads breaks the second subset of microorganisms. It may be that this second subset are the fungus and spores.
However, beat too much, and perhaps the DNA from the easy to lyse organisms is destroyed and this results in the low yields.
Lower Clay, High Carbon Soil
In this second panel are the results of the same experiment for a similar soil but with a high carbon content. The clay is still high (40%), but not as high as the first soil (45%) but the results are very different.
Here the yields of DNA from both garnet and glass beads start out even and then the garnet beads outperform the glass beads from speeds 3200-3500 RPM. However, we also see more shearing in the gel. This sheared DNA will be detected by the Nanodrop (and picogreen) as more DNA, so it can sometimes give a false sense of higher yields. This is why we always recommend checking the DNA on a gel and not just going by a spec readings.
For this particular soil, the overall yields are higher and we see that the garnet beads in this case give a very efficient extraction of DNA across a wide range of speeds that also peaks at 4200 RPM, after which yields do not continue to rise. This data also supports the idea that a maximum speed or time is reached where no further DNA will be extracted, and in fact, DNA may be lost.
1- These data show us how diverse and individual each and every soil is. A sample of beach sand is not going to extract like forest soil and clay soils will respond differently even between each other. The consistency of the soil, the level of biomass, and the organic content are going to influence how much DNA you have and the best way to homogenize.
2- It is important to always run an agarose gel to go with spec readings of the soil. Using the DNeasy PowerSoil Kit, only genomic DNA is isolated, not RNA. Other methods will isolate the total nucleic acid content including RNA, which will drive up spec readings and give a false sense of a good extraction. And for checking integrity, a gel picture will let you know if you are beating too hard or not hard enough.
3- Because every soil is different, it is always a good idea to do a preliminary test of your soil under a range of speeds or time and maybe even with two different bead types. As a starting point for the DNeasy PowerSoil Kit, we recommend 4000 RPM for 45 seconds, since this was in the optimal range for most of the soil types tested with both types of beads. However, you may find that you want to turn the speed down, or up, depending on whether your samples are high in spores, or low in biomass. A few test runs at some different speeds will let you know that you are getting maximal yields of high quality DNA.
As part of this study we collaborated with the Chris Kitts lab at Cal Polytechnic State University and his undergraduate students performed t-RFLP analysis on the DNA extracted from 5 soils using either the vortex or the PowerLyzer 24 Homogenizer and for garnet beads vs. glass beads.
In summary, we recommend that when starting a new project, do a speed-course study with your bead beater of choice to see if the same settings you used for your last soil still apply or if you need something optmized for the new soil. Since many of these samples are irreplaceable, a little extra time at the beginning may be worth the valuable data you will obtain later.