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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!

dan

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To start off with your last question, strandedness is something the reverse transcriptase does not care about. If you use random hexamers it will simply translate your -ssRNA into a + single stranded cDNA. Perhaps this diagram from Wikipedia makes things more clear, it is for an mRNA but the principle is the same:

enter image description here

This cDNA will then serve as a template for your primers in the qPCR which will turn it into a double stranded DNA molecule. In other words, after one round of amplication in a PCR reaction the DNA products of the + and the - RNA strand would be indistinguishable!

It therefore does not matter which strand you synthesise as DNA to make your standard curve. It will be double stranded so you just need to compensate for the other strand being a template in the PCR as well.

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  • $\begingroup$ First thanks for your answer. I thought that the RT would not care about strandedness, but I wasn't sure. Now, I am. Okay, for my example above you would say it doesn't matter whether I would synthesise the RNA as AUG or UAC? But what I didn't get is: What do you mean with 'I just meed to compensate for the other strand being a template in the PCR as well' ? Can't I use the synthetic +RNA dilution series as a standard curve for my unknown sample with -RNA? Sorry for my questions. $\endgroup$
    – Dan
    Oct 31, 2016 at 13:42
  • $\begingroup$ I was not aware you were going to make synthetic RNA as the template. It is certainly the most accurate but it also a lot more expensive to synthesise. Usually people use things like DNA gBLOCKs from IDT but I have no experience with ssRNA viruses and what the acceptable standard is there. If you do go for dsDNA then you need to keep in mind that there are two strands that can be bound by primers and you will underestimate the amount of ssRNA in your sample if you do not divide by two. $\endgroup$
    – mimat
    Oct 31, 2016 at 15:51

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