Dear all,
currently, I am investigating mouse stool samples on the presence of a certain mRNA from a certain species with 2-step RT-qPCR. The problem with fecal samples is that there are numerous bacterial species prevalent and I am trying to find the needle in a haystack. So far, my qPCRs show no template (huge Ct values, primer dimers via melt curves). I assume that my target cDNA is below the detection limit due to abundance in, for instance, cDNA transcribed from rRNA. People investigating microbiota using RT-qPCR either target a conserved protein region prevalent in several species or target 16S rRNA from one species. Both approaches imply more prevalent target mRNA in one samples than in my case.
Thanks a lot for you answer and the linked publications! Sample prep in this direction could be really helpful.
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