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?
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