Our team came back from the 4th Annual Argonne Soil Metagenomics Conference held in the Chicago area October 3-5 last week and a hot topic of discussion was filamentous fungi. Seems that ears were burning all over the cosmos as our technical support lines were also lighting up with questions about recommendations for isolation of DNA and RNA from filamentous fungi. So it looks like this tough critter deserves a blog post all its own.
Fungi are a funny breed of microbe. What other species can range in size from a single cell to the largest known organism on earth encompassing almost 4 square miles of soil (in the Blue Mountains of Oregon)? That’s right: Fungus. Fungus are everywhere, many beneficial, some tasty, and others deadly. No matter what you think of them, there is no denying that these are the toughest of microbes.
What makes them so tough?
What makes them so resilient to lysis? Why can they resist the same forces of heat and bead beating that would send most other microbes into a soupy mix of protons and neutrons? The composition of their cell walls is the key to their longevity. Fungi employ a combination of glucans with chitin to protect their cell membrane. Chitin is the same compound that forms exoskeletons in insects and when combined with calcium carbonate, makes up the shell of crustaceans. Pretty much, biological cement.
The composition of chitin is repeating units of modified nitrogen containing polysaccharides held together by covalent β-1,4 linkages. The structure allows for increased hydrogen bonding between polymers, generating chitin’s bionic strength. (see figure left)
Clearly this cell wall has been instrumental for the long term survival of fungi. A quick search on the evolution of fungi brings up much interesting reading, but to summarize in one sentence, the earliest appearance of fungi in fossils appears around the Proterozoic eon, 1,430 million years ago. Even fungi prove the earth is greater than 9000 years old!
On to the purpose of this post: to tell you how to get the most DNA and RNA from fungi. There are several approaches using MO BIO Kits you can take depending on the sample matrix.
DNA and RNA Isolation from a Pure Culture
For DNA isolation from a pure culture of fungus, we recommend using the glass bead tubes that come in the PowerLyzer UltraClean Microbial DNA Isolation Kit for mechanical homogenization. Ideally, a high powered bead beater should be used as well. The vortex will work but we usually recommend a heating step before the vortex step to enhance breakage. Try warming the cells in the bead tube containing lysis buffers at 65-70C for 15 minutes. If you have a high powered bead beater, such as the PowerLyzer or similar type instrument, you can skip the heating step but you’ll want to optimize for the best settings to not over homogenize.
For RNA isolation from a pure culture, try the UltraClean Microbial RNA Isolation Kit. This kit uses a silica carbide bead that has sharp edges which does a good job of not only lysing cells but shearing genomic DNA.
Some of our customers are using the PowerBiofilm DNA or RNA Kit with excellent results. Because of the rich polysaccharide nature of the chitinous cell wall, the chemistry in the PowerBiofilm Kit can help dissolve the glucans and make the cell wall easier to break. And the bead tubes in the PowerBiofilm kits have a combination of bead sizes which may help break down more of the cells, especially in a culture with mycelium. And of course environmental samples containing inhibitors will be removed with the PowerBiofilm Kits.
Fungal DNA and RNA Isolation from Soil
When it comes to soil and fungal microbiomes, we would recommend using the PowerLyzer PowerSoil DNA Isolation Kit because of the glass bead tube. The glass will work better in the high powered bead beaters. Read more on the comparison of bead types and soil DNA isolation here.
For RNA from soil, the best product to use is the RNA PowerSoil Isolation Kit. This kit combines the silica carbide beads from the Microbial RNA Kit with a phenol based lysis to ensure that all microbes have no chance of staying intact. And since this product starts with 2 grams of soil, you’ll get enough RNA from even low biomass samples. For tips and tricks using this kit, check here.
Water Filter Membranes
And of course, if you are working with water samples and 47 mm filter membranes, then the PowerWater DNA and RNA Kits are the best choices.
As you can see, the matrix is equally as important as the target organism.
Other Sample Types:
We could go on and on with sample types: saliva, body fluids, blood cultures, swabs, stool…. etc. The basic answer is, if you have a biological sample and need fungal RNA or DNA from it, we have a recommended method for it.
Let us know whenever you need help and we’ll tell you the best kits to try and then set you up with samples too.
There are so many interesting and vitally important fungi in need of further study, and MO BIO Labs has the tools to do it!