I want to standardize RT-PCR for various microbial groups. Is it possible to do so using metagenomic DNA which might contain the same groups? Or do I need to use DNA extracted from an axenic culture of respective group?
Hi ! I don't think it's possible because to standardize you have to quantify the DNA concentration of each group separately, and you can't do this with a metagenomic sample. An axenic culture of respective group might be a better choice to quantify a first concentration range for your standard curve.
Hi ! I don't think it's possible because to standardize you have to quantify the DNA concentration of each group separately, and you can't do this with a metagenomic sample. An axenic culture of respective group might be a better choice to quantify a first concentration range for your standard curve.
No, you can not use metagenomic sample. Unless, you know from some other sources (which is highly unlikely) exactly how much DNA in your metagenomic sample is represented by each targeted group...
However, I also don't think that you need an axenic culture per se. If you have mixture of very closely related strains or even species, DNA of which will produce the signal in RT-PCR, I think, you can use such mixture without going through the pain of axenic culture prep.
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