Hello all,
I want to perform qRT-PCR to determine the levels of bacteria in infected tissue biopsies. We do usually isolate RNA via the trizol-phenol-chlorophorm method. In these biopsies, the mentioned protocol will provide us both bacterial and eukaryiotic RNAs.
I have read few people stating there is no need to separate thes RNAs for developing PCR. This would be nice for us since it will allow us evaluate both procaryotic and eukaryotic mRNA expression in the biopsies. But I would like to double ckeck this information before handling the tissue samples.
So, my questions are:
- Is it necessary to remove the eukaryiotic RNA from these samples? Would these molecules interfere in my bacterial mRNA evaluation?
- In case we should remove the eukaryotic RNA: Do you know any in-house protocol, instead of a commercial kit?
Thank you so much in advance! ;-)
- In essence, if your primers are species specific; that is specific to your bacterial orthologous gene then no the presence of Eukaryotic mRNA should have no effect on your analysis
- The key will be good species speciifc primer design
- use therefore of primers designed to your mRNA of interest with species specfic accession number and having made the primers then subject them to BLAST analysis to esnsure they only bind to your bacterial target is key
Hi Barbara,
I agree with Prof Dawkins-Hall. By designing specific primers, you can achieve your objective using total RNA as template.
I just want to add here that, in some case, especially in low level viral or bacterial RNA detection, highly abundant host ribosomal RNA may interfere. in that situation you may consider using a host sepcific ribosomal RNA depletion method. However, such kits are commercially available and I am not sure if any in-house protocol is available or not.
best wishes,
SD
Hi,
agreed with the comments above. however, it seems you would rather like to know the abundance of bacteria. In the case you still have tissue I would rather propose to use DNA and to amplify 16S genes. In this regard you can better determine the bacterial load, as I don't know how you would like to do this with RNA.
Best Sven
Thank you all for your useful comments!
As you said, I thought eukaryotic RNA should not be a problem if we design nice specific primers for bacteria.
The point is that I would like to use the eukaryotic RNA as well to evaluate whether the presence of bacteria could affect other markers from the host tissue, that is one reason why I would be interested in keeping the whole RNA pool. If this was not feasible, I could divide the biopsies into 2 pieces and proceed independently.
Regarding the DNA isolation, this is a good advice to discuss with my colleages.
Many thanks again ;-)
Greetings from Spain!
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