Question

Active microbial community diversity based on cdna

Answer 1

Let start with the basic; DNA and RNA are relative stable molecules. In nature, they are degraded mainly due to enzymatic catalysis. Of course, RNA is less stable than DNA in oxic conditions, but this is not the case here. Microbial communities in cold soils are dominated by psychrophiles which are able to have an active metabolism in those environments. Investigation of microbial communities via 16S amplicon sequencing in DNA level will show you who is present, where in RNA level will show you who is active there. This is a well known process and people doing that for long. The difficulties lying in the following:
1. Be able to stabilize your samples as soon as possible and not having community shifts during sampling and prior of extraction. Several ways to do so; Liquid nitrogen, RNA protect or Lysis buffer in situ. Since the environment is cold, and liquid nitrogen difficult to transport, I would go either with RNA protect or lysing buffer directly (and then freeze). Actually, this is how I am collecting environmental samples for RNA analysis. I used this procedure to collect permafrost RNA samples.
2. How much RNA you can extract from your samples. There is a huge discussion among experts, and dozens of protocols. Experience taught me that there is no universal protocol and if you want to evaluate a protocol you need to do it with the actual samples! This, sometimes is very difficult. In principle you need to keep in mind the following principle: harsh conditions to break the cells and release nucleic acids, not too harsh because you will co-extract several inhibitors (i.e. humic acids) and you might degrade your RNA.