As part of our research on the best practices for soil microbial DNA extraction, we collected a wide variety of samples for product development. Therefore, when we were developing the protocols for the PowerLyzer 24 Homogenizer, we developed a protocol that is applicable for the majority of the samples tested. Our previous work on homogenization and bead tubes showed that some bead types could result in an increased yield of DNA, depending on the soil type. We decided to do a similar study using the PowerLyzer 24 Homogenizer to determine if DNA yields and integrity with high-powered bead beating differed between two different soil types, using identical protocols. It is not uncommon for researchers to simply adopt a protocol from a published journal article for their soil type without optimizing the conditions. However, does one protocol really work best for every soil type?
Here, we share with you our results that answer this question. We compared the results of DNA yields and integrity from two different soil types; one with high clay content and one with high carbon content. High-powered bead beating (using the PowerLyzer 24 Homogenizer) was used with two different bead types; 0.1 mm glass beads (cat. no. 13118) vs. 0.7 mm garnet beads (cat. no. 13123). The results were very surprising.
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 increased the speed all the way to 5000 rpm, the highest setting on the PowerLyzer 24 Homogenizer. All preps were run for 45 seconds.
DNA was isolated using the DNeasy PowerSoil Kit, quantified on a Nanodrop™ and run on gels to check for integrity. We plotted the yields on bar graphs (Figure 1).
High clay, low carbon soil
This first figure shows the results of the higher clay soil with the PowerLyzer 24 Homogenizer.
Figure 1. Low carbon content clay soil DNA yields on the PowerLyzer 24 Homogenizer. Lane M: DNA marker.
The glass beads, in general, extract more DNA from this soil type and this extraction peaks at a speed of 3900–4200 rpm (lanes 14 and 16). Interestingly, when speeds higher than 4200 rpm are used, yields decrease. This demonstrates that you can beat a soil too much or too hard and lose DNA.
Alternatively, at speeds slower than 3900 rpm, the glass and garnet beads were extracting equal yields. Why could this be? One theory is that perhaps the soils have two predominate 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. Then, the boost at 3900 rpm with the glass beads breaks the second subset of microorganisms. It may be that this second subset is the fungus and spores. However, if the sample is beat too much, then perhaps the DNA from the easy to lyse organisms is destroyed and this results in low yields.
Lower clay, high carbon soil
The second figure contains results of the same experiment for the soil type with high carbon content. The clay content is still high (40%), although not as high as the first soil (45%), but the results are very different.
Figure 2. High carbon content clay soil DNA yields on the PowerLyzer 24 Homogenizer. Lane M: DNA marker.
The yields of DNA from both garnet and glass beads begin even and then from speeds 3200–3500 rpm, the garnet beads outperform the glass beads. However, there is also more shearing in the gel. This sheared DNA is detected by the Nanodrop (and picogreen) as more DNA, so it can sometimes give a false sense of higher yields. Therefore, we always recommend checking the DNA on a gel and not depending solely on spectrophotometer readings.
For this soil type, the overall yields are higher and the garnet beads give a very efficient extraction of DNA across a wide range of speeds. This peaks at 4200 rpm, after which yields do not continue to rise. This data supports the idea that after a maximum speed or time is reached, no further DNA will be extracted, and in fact, DNA may be lost.
• Variation in sample type can affect DNA yield
• DNA integrity should be checked on an agarose gel
• A range of speeds, time and bead types should be tested
First, as shown by the data, variation in sample type can affect DNA yield. Different clay soil sample types result in differing results. The consistency of the soil, the level of biomass and the organic content can influence the type of homogenization protocol that needs to be used and the DNA yield.
Second, it is important to always run an agarose gel to compliment the spectrophotometer readings of the soil. With the DNeasy PowerSoil Kit, only genomic DNA is isolated, not RNA. Other methods that isolate the total nucleic acid content including RNA will increase spectrophotometer readings and provide a false sense of a good extraction. In addition, a gel picture enables the assessment of DNA integrity, so that you know if you are beating too hard or not hard enough.
Third, due to soil variation, it is always a good idea to do a preliminary test of varying soil samples under a range of speeds or time and possibly even with two different bead types. As a starting point for the DNeasy PowerSoil Kit, we recommend 4000 rpm for 45 seconds. This was in the optimal range for most of the soil types we 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 different speeds will let you know whether you are obtaining maximal yields of high-quality DNA.
As part of this study we collaborated with the Chris Kitts lab at California Polytechnic State University. His undergraduate students performed t-RFLP analysis on the DNA extracted from 5 soils using either a vortex or the PowerLyzer 24 Homogenizer, and garnet beads vs. glass beads.
In summary, we recommend that when starting a new project, conduct 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 optimized 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.
Looking for tips to improve your RNA isolation from plants, animal tissues and cells? Check out our earlier post.
Trademarks: NanoDrop™ is a trademark of Thermo Scientific.
Authors: This post was originally published on mobio.com, and has been updated and modified by Heather Martinez and Miranda Hanson-Baseler.