I have recently come across some papers that suggest 16S RNA sequencing as a technique to know the active microbial population in metagenome and require some views about it. Also if this experiment is effective, I would like to know how to specifically isolate rRNA as most kits offer specific isolations of mRNA only.
Hi, I did 16S rRNA sequencing from metagenome sample previously. What I did was to extract the metagenome DNA using conventional method. I sent the DNA to a sequencing company and they did the NGS using primers that amplify the 16S rRNA region of the microbes.
Yes, you are right. To understand bacterial diversity, we extract metagenomic DNA and send for NGS. But this technique did not give information about how many microorganisms are active and dormant.
So there are some papers where scientist extract rRNA from the samples and did NGS which generate information about active and dormant bacteria in the sample.
So with the help of NGS we know that wat is the diversity of bacteria present (at phylum or genus level) in a given sample but we don't know how many are dormant and active.
we want to know the technique to extract rRNA from the plant sample.
Any extraction protocol to extract total RNA, if no specific treatment is included to avoid rRNA, will actually extract mainly rRNA (over 90% of total RNA) and minor amounts of mRNA. RNA strands is in general a very fragile and unstable molecules, so I recommend you to proceed to produce cDNA as soon as you obtain the RNA. There are many possibilities with bacterial RNA but one is to use random hexamers or external primers comprising the targeted region for the PCR amplicon generation (like nested PCR fragments). As example you can use for reverse transcription primers of the outer conserved regions of 16S (like 17F and 1492R) and then for PCR, use on this cDNA template, primers targeting regions inside those fragments (such as V4). Or you can use for the RT reaction random hexamers (NNNNN) and use that cDNA produced as template for PCR amplification of region V4.
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