I'm looking at RNA-based rRNA to ID soil microbes. Using MoBio RNeasy kit to isolate RNA from soil. Looking for a compatible kit/process to convert the RNA to cDNA for analysis. I'm new to using RNA-based community analysis methods, and will be snap freezing my samples in the field to bring in for lab analysis. Any field tips are welcome!
Hi Ali,
There are basically 3 approaches for reverse transcription. In principle, they only differ in the primers used, so each can be done with the same kit. You can use gene specific primers (this is best in your case), random hexamers (RTranscripting all RNAs, but resulting in fragments), and oligo(dT) primers (for eukaryotic mRNAs, with polyA tails).
For bacterial identification, you should use gene specific primers for the 16S region. Check out some publications with samples similars to yours, most probably there are primer pairs that were already tested.
You can check out this tutorial for more information on what should you take care of during the process:
https://www.thermofisher.com/hu/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/rt-education/reverse-transcription-setup.html
As for the kit, if your product is clean, there should be no compatibility issues. As you used MoBio for isolation, you can use e.g. Qiagen (which is owned by MoBio) One-step RT PCR kit. But really, any RT-PCR kit should work.
Thanks! I've done DNA analysis before, and was worried the RNA might decay during transport before I could extract and reverse-transcript it. thanks for the kit tips!
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