I would like to perform absolute quantification of RNA viruses in plant tissue and was wondering if approaching this through in vitro transcription makes sense.
I don't understand why do you want to "in vitro" transcribe DNA to RNA? You already have your plasmid with your control sequence that you can quantify accuratelly. Furthermore, to perform qPCR you will have to reverse-transcribe yor RNA to DNA!!
Here is a good article about using RNA as standards in qPCR:
http://www.biomedcentral.com/1471-2164/12/118
There always lingers the question, however, whether the standards you prepare amplify at the same efficiency as your biological sample extracts do. So bear this in mind and account for it mathematically if this disparity presents itself.
Rudolph,
The question you ask is a great one - and some of the answers here already touch on some of the issues at hand. But, like I mentioned in a prior post above, whatever you decide to use for your absolute standard, cloned sequence in a plasmid, RNA made from that, purified amplicon etc., you will invariably find that the sample material (plant extract with virus in it), will amplify the viral signal at a different efficiency than the absolute standard will. So, it is indeed a legitimate approach to just use your plasmid (as Junaid suggests above) - but, that is not the end of the story. You must then reconcile the two different efficiencies that you will see between the absolute standard curve and a standard curve made from virus-infected plant extract material (even though the same primers are being used and the same target is being amplified). It is the context in which the target is presented to the qPCR that causes this difference in efficiency of amplification. The publication attached here demonstrates this difference quite well.
The way to calculate copies of virus in your biological extracts using an absolute standard curve made of another "material" is summarized in the attachment in my next post (below). I hope this helps...
Here is the equation (attachment here) you can use to reconcile the two semi-different universes of biological material extract and 'absolute' standard material standards...
To use the equation you first need two different standard curves:
1.) The absolute standard curve (e.g. made of serially-diluted plasmid with sequence insert), and
2.) A standard curve made from virus-infected plant material.
You use each standard curve to estimate efficiency of amplification in each case.
The absolute curve will give you Eamp of 1.97 (i.e.)
while the plant extract curve will give you an Eamp of 1.88 (i.e.)
You will use the y-intercept (the Cq value for 1 copy) of the absolute standard curve in the equation as well. These values (along with arithmetically averaged replicate experimental unknown Cq values) are plugged into the equation (attachment) to apprehend the estimate of initial # of copies of virus per qPCReaction for your biological samples. Knowing the # of initial copies in each reaction then allows you to back-calculate to copies per mg of plant material extracted - all while taking into account the differences in efficiency between biological sample and absolute standard material to do this. Ideally, using uninfected plant extract spiked with known copies of absolute standard would obviate the need for the 'pure' absolute standard curve mentioned above, since, what you really want to know in the first place is the efficiency of amplification of your target within the matrix of the biological material you actually extract. The equation then simplifies merely to:
copies per reaction (Xo) = Eamp^(b-Cq).
where: Eamp = 10^(-1/slope), and b is the y-intercept of the (e.g.) plasmid-spiked plant material extract standard curve. The Cq value is the observed arithmetically-averaged replicate Cq values generated from each of your experimental unknowns.
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