Regarding isolation procedure you can use promega RNA isolation kit. We generally use promega kit and they offer quite good isolation of RNA. 

If you are concerning about observing diversity of the microbial isolates then you can use DNA isolation kit. First you have to isolate the DNA and to amplify the 16S rRNA you will be needing universal primers that are available through different companies. The selection of universal primer also depends upon the nature of bacteria and fungus that you are isolating.We generally order and use the universal primer from Macrogen korea (you can google the macrogen website to view different primers)and perform sequencing of  the region to get 16 S sequence. After that its all about bioinformatics tools.

I have also done similar works on sequencing but its on thermophiles. You can download my paper from research gate and hopefully it will help you.

GOOD LUCK

Hi Ximena

You can try the RNA Powersoil kit by MoBio. It is pretty expensive, however. If you need to save and do a customized RNA extraction, try the one from this paper:

A sequential co-extraction method for DNA, RNA and protein recovery from soil for future system-based approaches. (DOI: 10.1016/j.mimet.2014.06.004)

I have used it for DNA extraction, and I always get a whole lot of RNA too. As far as separating 16S and 18S rRNA, these are very similar in size, so electrophoretic separation and subsequent gel clean-up is not really an option. I would use bacterial 16S-specific primers to amplify a section of the 16S rRNA gene from your cDNA after reverse transcriptase PCR. As far as functional analysis goes, you can also look into nifH and cbblR to look more specifically at nitrogen fixation and carbon fixation, respectively.

Good luck! 

Thanks! I'd like to give some more details... I'm planning on doing DNA analyses for 16S and ITS for bacteria and fungus, respectively. That's why I want to separate 16s and 18s from the soil RNA, so then I can sequence the remaining RNA as a proxy of the soil community functioning.

You can always do a PyCrust functional prediction based on which taxa were identified from the amplicon sequencing to get an idea of what to expect from your RNA sequencing. You could remove rRNA sequences from your dataset by using a database-based filtering approach. If you want to avoid sequencing the rRNA genes altogether, you could consider doing a rRNA subtraction with probes (see attached paper).

Hope this at least points you in the right direction!

Kind regards,

Riegardt Johnson

MO-BIO power soil RNA extraction kit would be great I used for one of my transcriptomics study.

Dears, do you know the protocol for promega RNA extraction kit? Is the standard one? The Qiagen RNAeasy es rather expensive and I have lot of soil samples for RNA extraction. Kind regards.

Marco A. Campos see in the Material and Methods.