Standard curve from plasmid - for cDNA quantification (Aug/06/2008 )

Hi all,
I've been asked a question that I'm not sure how to respond, so here I am looking for help
I want to quantify viral RNA by RT-qPCR, so my target is cDNA. However, I've made my standard curve using a plasmid that containg my target gene, ie: the standard is dsDNA while my sample is ssDNA... Will this affect my calculations, and does anyone know how?
If the answer, as I'm afraid, is yes, how do I get round it.

Thanks very much in advance.

Hi all,
I've been asked a question that I'm not sure how to respond, so here I am looking for help
I want to quantify viral RNA by RT-qPCR, so my target is cDNA. However, I've made my standard curve using a plasmid that containg my target gene, ie: the standard is dsDNA while my sample is ssDNA... Will this affect my calculations, and does anyone know how?
If the answer, as I'm afraid, is yes, how do I get round it.

Thanks very much in advance.

if you want to do absolute quantification, you need an RNA standard which you reverse transcribe like your samples. you cannot use DNA because there is no control for the efficiency of the reverse transcription step.

I agree with Ned Land. You could try and fudge it and say that your standards are equivalent (so 1 uM standard would be 2 uM of ssDNA) to your cDNA, but it isn't correct.

Basically if you are using a standard curve method for absolute quantification you need to use known quantities of RNA starting material. IVT your GOI and reverse transcribe a series of known dilutions (keeping the total RNA quantity the same as your samples by adding yeast tRNA). Total RNA concentration consistancy is very important, otherwise the amount of cDNA for your GOI will not be directly proportional to your dilution factor. See Stahlbert et al. (2004) in Clinical Chemistry - Properties of the reverse transcription reaction in mRNA quantification.

HTH

hi mates,

What i have done in my last Qpcr, i used a different dilutions from the orignal plasmid that i have used for transfection.

I don't know if this the right way?

Hello again everyone. First of all thanks very much for your answers they pretty much confirm what I knew which is always reassuring

Second, another question: Is there anyway round this issue if I cannot make my standards from RNA? ? ?

I used to do qPCR on mRNA extracted from cells, and used a pool of all my samples as a standard curve, it was never absolute quantitation but arbitrary units, kind of a combination between absolute quantitation and delta-delta-Ct

However these days I'm purifying RNA from genetically engineered viral particles that only contain GFP, so I thought a plasmid containing GFP will be good enough for standard curve. I could make some RNA in vitro transcribing from my plasmid, however my resources are limited and justifying the expenses might be quite difficult, which is why I’ve been looking for alternative choices already available for me.
Before my post turns into a rant which will better fit in a different topic in this forum I should stop here.

Thanks again to everyone, any more suggestions will be more than appreciated.

Have a look at

Rutledge and Stewart 2008. A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCR. BMC Biotechnology 8:47