I need to do total nucleic extraction from fungi out of clinical samples. At the moment we are using bead beating followed by a total nucleic extraction protocol on the Roche MagNA pure compact. Quantification of spores going into the extraction (can't really quantify mycelia but have tried those too) and subsequent PCR (with a ribosomal target) suggests we are getting a good yield of DNA from this procedure. However, when we do reverse transcription PCR to pick up the RNA in the ribosomes as a target too. We are not seeing much more of a PCR product. Is there a way to ensure ribosomal RNA gets extracted in total nucleic extraction protocol.?
Hi, you should be able to visualize the rRNA either after agarose gel electrophoresis or PAGE. There is usually so much rRNA in the cells that they tend to form 3 prominent bands.
Maybe you are loading too much template or there is something inhibiting the RT reaction that you carry over with the template, so I would first try the RT PCR with lower amount of template.
Thanks Jiri. I'm not sure too much template/extract is the problem. We've diluted it right down to where all amplification ends and we just get the same amplification curve for with or without RT step.
You should first check the integrity of your RNA i.e. on agarose gel you should obtain intact bands for rRNA (3 prominent bands where the top two should be more intense than the lower one). If you see smears in the gel then your RNA is probably degradng hence you are not getting RT-PCR. But If that is fine and you are also using the same primer and buffers for both your normal DNA PCR and RT-PCR, then the only thing is that the RT step is not working. Are you incorporating a 65 degrees melting step before you incubate with RT? this removes secondary structures that can inhibit your RT reaction.
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