I'm really new to RT-qPCR and probably I've just a minor problem than I think.
So, what's my problem about.
I want to quantify a virus by a specific area within a gene on its genome. I've specific primer which are working fine with the extracted virus rna from my samples. But now I need a template for a dilution series to quantify this virus. And there's my little problem. And maybe you can help me.
So I know that the genome of my virus is a negative sense single stranded RNA, so -ssRNA, and moreover I know the virus strain and the gene, which RNA I want to synthesize for the quantification. So I looked it up on ncib and I found the sequence of the gene, but it is listed as the complete cds of the gene and not the RNA sequence. And as I'm right the cds is the same sequence as the positive sense RNA, with Us instead of the Ts?!? So is it right for synthesizing, that I have to translate the cds first in +ssRNA and then in -ssRNA and this -ssRNA sequence I have to synthesize to get a standard?
If the cds would be: ATG , the +ssRNA would be: AUG and therefore the -ssRNA would be: UAC and this UAC , I would have to synthesize? Sorry for this stupid question but I am a lil bit confused at the moment with the difference of - and + ssRNA regardind the RT-qPCR. Another question would be, whether the Reverse transcriptase with Random hexamer primer is specific for -ssRNA or +ssRNA. It isn't ? So my quantification would also work with the AUG as a template for a standard curve?
Thanks a lot!