My lab is starting to use RNA-seq to characterize gene transcription changes in the gut microbiome, and I am wondering if it is at all feasible to assess transcripts using RT-qPCR? We are trying to assess changes in mRNA levels independent of species, and there are a couple problems were encountering.
For one, there is too much sequence divergence across species to use a single primer. So we have tried to design primers that work for individual phyla, but even then it appears as though some species are more well suited towards the primer than others.
The second problem is finding a suitable reference for quantification. There's no way were going to make plasmids for 100+ genes, but I don't know if any small number of plasmids would be representative of overall mRNA levels.
Can anyone tell me whether they have thought about this before or successfully done this? Is it easier/harder than I realize? Anything I need to know about before I continue?
Of course is possible to perform RT-qPCR from gut microbiome. However, you are trying to measure the gene expression of several genes for several species at the same time. So I suggest to apply a metagenomics analysis to determine which species are your sample and determine the relative abundance for any specie of interest to focus the design of specific primers to determine the expression levels for the genes of interest for specific species in your samples.
Regards
Hi Aaron, just a bit belated, but thanks for the response.
Wanted to provide an update in case anyone is looking at this question.
Practically, designing primers for RTqPCR from microbial communities is real tough, even for an individual species, taking into consideration strain level sequence variability.
I think you made a really good point. Ideally, you would sequence all the genes you are interested in for each individual sample, then try and design primers for any of the sequences you identify.
Practically, my advice would be to stick to an individual strain, and ensure your reference and target genes have no off-targets on other strains. But still be prepared to find sequences that are slightly divergent from currently indexed strains.
If anyone finds a better way to approach this without using RNA-seq please keep me posted.
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