Dear all,
I’m working on construction of cRNA standard for RT-qPCR by in vitro transcription from PCR template. This standard has identical gene sequence as a target gene. I designed specific primers with T7 promotor sequence at 5’-end on forward primer. For transcription I use MAXIscript™ T7 Transcription Kit (ThermoFisher). I used PCR template for in vitro transcription, then I precipitated cRNA by ammonium acetate. I used precipitated cRNA as a template for RT-qPCR with the same primers, but without the T7 promotor sequence. I compared RT-qPCR run with qPCR to determine an amount of DNA contained in the sample. There wasn’t any difference between these runs, the sample contained only DNA. In vitro transcription failed and I can’t find the reason why. I used PCR mastermix with polyA overhangs, is it wrong? Or is it better to use the reverse primer with oligo-dT at 3’-end? Is it good idea to use this method for the cRNA construction? Does anyone have prior successful experience with this? Many thanks in advance!
I don't really understand what you're doing (do you make your cRNA into cDNA at any stage? This is not mentioned), nor do I think what you are doing is necessary.
If all you want is a standard, so you can measure transcript numbers rather than transcript relative amounts, just...use a PCR product. Run a PCR with the same primers you're going to use in qPCR, run the product on a gel and cut it out (to remove primer dimers), purify it, determine the concentration via spectrophotometry and thus calculate your molecules per ul, and use this as your standard, at dilutions from perhaps 10^7 molecules per well down to 10^0 or 10^-1 (10-fold serial dilution is usually fine).
Remember, PCR is an exponential process, so the vast majority of amplifications that will occur in your reaction will be using PCR products as template, not cDNA:
Cycle1: 10 molecules of cDNA template, no PCR products
Cycle2: 10 molecules of cDNA template, 10 PCR products
Cycle3: 10 molecules of cDNA template, 30 PCR products
Cycle4: 10 molecules of cDNA template, 70 PCR products
Cycle5: 10 molecules of cDNA template, 150 PCR products
...
Cycle20: 10 molecules of cDNA template, 5242870 PCR products
and so on.
John Hildyard
Thank you very much for your quick response. Do you think it is possible to use just the PCR product when I need to get the standard curve for RT-qPCR? I need to quantify RNA samples and there would be no reverse transcription step if I used the DNA in the standard curve. I just wanted to have the same conditions for samples I quantify and for standards in one run.
Preparing cRNA and then reverse transcribing it is wholly unnecessary, and indeed counterproductive. Reverse transcription cannot be considered to be a perfect 1:1 process, so it will always introduce some noise taking RNA to cDNA, but you cannot measure noise by introducing additional noise of your own.
Using a PCR product as a standard curve will tell you how many transcripts you have in your cDNA: while this number may not be exactly the same as your original mRNA copies, it is at least a number.
Using a reverse transcribed cRNA as a standard curve will tell you (maybe) how many transcripts you have in your cDNA, but will now be subject to cDNA synthesis noise from your extracted RNA and your cRNA, with no easy way to discern which noise is which.
The assumption in cDNA synthesis is that it is always 1:1. This is probably not true, but it is also probably not hugely incorrect. And it is also generally considered to be non-biased, in the sense that it does not favour specific transcripts over others: if your cDNA synthesis is only 60% efficient, it will be uniformly 60% efficient, so you gene of interest will be reduced by exactly the same amount as your reference genes.
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