Hi everyone,
I am in the very initial stages of planning an RNA sequencing experiment. I want to find out the differential gene expression in my bacterial strain of interest when exposed to different test conditions compared to a control condition. I know that this bacteria has a plasmid, but I do not know its copy number and I also do not know whether the copy number varies between test and control conditions. I am concerned that if I see plasmid genes upregulated in the RNA seq experiment, it might be due to (potential) differences in copy number. Is there any way to find out the copy number of plasmid from the RNA seq data or would I have to do a qPCR for that purpose? Also, if copy number does differ between test and control conditions, is there a way to integrate that information when interpreting the RNA seq data?
Thanks,
Yoana
In my opinion, qPCR would be safe. RNA-Seq data would provide some insights (e.g., up-regulation of all genes belonging to a specific plasmid likely to be from greater PCN) but qPCR will provide definitive answer. In general, it's also recommended to conduct some qPCR on selective genes to confirm RNA-Seq data.
Hello
I recommend from the same cells you extracting RNA for your RNAseq analysis, you do extract DNA at the same time. Then you can quantify the plasmid copy number by a qPCR, then you would consider a normalization procedure (very specific) for your expression of plasmid genes.
All the best
Yoana Petrova , are you looking at the gene activity of the bacterial genome or its plasmid gene activity? In other words,are there specific target genes in your study?
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