I am trying to do the InVitro Transcript, but it's not working for me. I have only 2 months remaining for my research in the USA. Can I try something else like cDNA or total RNA to draw the Std Curve, so I can start my quantification experiments?
OneStep RT-qPCR with known cDNA standards, is a good way to do it well, but I dont fully understand your problem. I am doing gene-expression and miRNA analysis with this tech.
RNA prepared from dilutions of a viral culture supernatant known titer can also be used for preparation of cDNA; obviously, the RNA present in each sample should be quantified before cDNA synthesis. We have achieved good relationship between these controls and cloned products in our laboratory.
Total RNA extracted from the infected tissue works like a charm for us here. We carefully TRIzol-extract bits of lung, isolate the RNA, then use it in One-Step RT-qPCR (meaning that the RT and Taq enzymes are both present in the mastermix). We use hydrolysis probe and Fwd and Rev primers. We find our virus every time and use the general rule that ~37.5 = 1 copy if reactions are nearly 100% efficient (there are more complexities to that calculation - but, to save time, that's all I'll say). Inhibition lets up after RNA is diluted to at least about 1:340 by the time it sits in-well. Standard curves can go from 1:350 to about 1:10,000 typically, and all unknowns are happy being diluted somewhere between 1:500 and 1:800 -- whatever works best for you. For the individually-assessed samples, our in-well total RNA concentration for each is 0.7844 ng total DNase-treated RNA/uL. If you fail to dilute your samples enough, you can miss detecting your virus entirely on account of RT and/or Taq inhibition by gunk (!) in your samples. But, loading the same amount of nucleic acid per well is an absolute must these days for those who wish to perform sound qPCR/RT-qPCR. Hope this helps~!
My aim to absolute quantification of virus inside single aphid (small Insect) after different time period of acquisition (intake of virus from plant) using the TaqMan probe. I was trying to draw the Standard Curve. For that I am trying to make the In Vitro transcript of my Virus as it is RNA virus. My Question is that, Is In Vitro Transcript is necessary to draw the standard curve or we can use the plasmid or cDNA as standard?
The point you bring up is always tricky to address - but here's the way I try to explain this:
The context in which the target is presented to the qPCR will yield different efficiencies of amplification for that same target...
Whether:
Target A is presented as gDNA
Target A is presented as ds amplicon
Target A is presented as ss cDNA
Target A is presented as super-coiled double-stranded plasmid DNA
Target A is presented as linearized plasmid DNA...
Target A is presented as plasmid RNA
Target A is an in vitro transcript...
And also, for the above list, whether or not they were isolated from a
biological source or not will also affect performance/efficiency.
E.g., if you use purified in vitro transcript as the standard to evaluate virus isolated from your plant material, you still will need to know the efficiency of amplification of virus isolated from your plant material (e.g. do a serial dilution curve of infected plan material) - the biological extract of target will not perform the same as your purified standard curve material!
All contexts of Target A in the above list will have the tendency to amplify at different efficiencies using the same primers/primers-probe for the very SAME target; all on account of target "context" ... i.e., the different molecular geometries presented to the qPCR as "target" affect the initial qPCR kinetic directly for that target.
To confound things further:
http://www.sciencedirect.com/science/article/pii/S1046202312002290
A.K.A. "qPCR efficiency is not understood fully yet."
Name what you use, and use what you name; is the name of the game.
One context of a target does not confer kinetically identical behavior to the other possible contexts of that same target. Essentially, qPCR/RT-qPCR efficiency is not to be thought of as a 'constant' for all possible contexts of the same target. Efficiency is context-dependent.
So, we can only measure a facsimile of efficiency of any target depending on what costume it arrives in.
In your case, if you do have access to a readily-quantifiable in vitro transcript, and you can extract that quantifiable transcript the same way you've extracted your infected plant samples, you could then use it as a fairly reasonable standard that would allow you to compare and measure things on the right basis.
Your other option is to go ahead and use the cDNA approach - and merely report what you did. Stay in-costume as much as possible throughout.
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