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...
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