this will lead you to get gene expression of prokaryote and eukaryote if you need for your work

Yes, I know, but I would like to be sure I can still do a reliable community analysis based on amplicon ribosomal sequencing, from cDNA coming from total RNA extraction from my sediment samples. It would be good also if after detecting representative samples, we could sequence the mRNAs present to get insight on the gene expression.

Yes, it's possible, for example.

http://aem.asm.org/content/80/18/5761.full

This paper does shotgun metatrascriptomic, but there are other papers out there that do amplicon based studies. Just search for them.

Yes you can do amplicon sequencing from cDNA. Just treat cDNA as gDNA and set up your pcr with the same primers as you do for gDNA. 

Thank you so much!!!! now I need to find financial support to be able to buy the total RNA extraction kits for soil and then sequencing, as I asked for a quotation for amplicon sequencing with 250 samples and it seems far beyond what we could get from a grant :(

You can try Trizol method for total RNA extraction. Kits for cDNA synthesis are inexpensive. With illumina, sequencing is relatively very low.

Dear Susana,

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. 

If you need more information how you can do it, limitations and amlpicon RNA seq strategies, please contact me directly.

Hope this helps

Best,

M