Julien is clearly wrong. Two genes having the same Ct do NOT need to have the same concentrations (copy numbers)!
Stephan is right, but it requires that you know how many genomes you effectively introduced for quantification. It might be adviseable to measure another gene's copy number (with already known copy number) for reference.
Artur is absolutely correct. You need to read the literature. Here is one more paper:
Clinical Chemistry 02/2003; 49(2):219-29.
You have two ways. If you know a gene which is represented in a single copy in your genome of interest you can use this gene as reference. Because for each PCR cycle you multiply by 2 the number of PCR product copy, you can deduce the copy number of your gene of interest (GOI). For exemple, the gene of reference (single copy) present a Ct (or Cp) at 25, your gene of interest has a Ct at 24 cycles, suggest that the copy number of your GOI is 2. It is right only if the efficiency of your PCR is 100% (you must check it before)
If you don't know any gene of reference which is single copy you can do a relative estimation. You can find a method in the paper in attached file.
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To obtain a copy number, you need to clone your PCR fragment. Using this plasmid, you can calculate a number of molecules (and not number of mol....). In your PCR plate, you put a standard curve, from 10, 100, 1000, ... copy of your plasmid with the DNA fragment, and your sample.
For the genomic DNA, it is very useful as you can calculate precisely the number of copy per genome.
It is nicely explained in the attached Nature paper please go through it
Julien is clearly wrong. Two genes having the same Ct do NOT need to have the same concentrations (copy numbers)!
Stephan is right, but it requires that you know how many genomes you effectively introduced for quantification. It might be adviseable to measure another gene's copy number (with already known copy number) for reference.
Artur is absolutely correct. You need to read the literature. Here is one more paper:
Clinical Chemistry 02/2003; 49(2):219-29.
Hi Jochen, I understand the need for absolute quantification by calculating the effective number of genomes introduced. But how is Julien wrong, he suggested the use of known single copy gene as a reference. So relative quantification is possible right? May be it would be better to use multiple known single copy references for better resolution, but I do not get what is wrong with this approach of relative quantification if both the primer pairs have similar or same amplification efficiency, are very specific for the GOIs. The problem I have observed with using a plasmid dilution series for calculating a standard dose response is that using different templates the amplification efficiency can be different for primers so again it brings in another problem where it addresses one. What are your thoughts on this?
Hi Amey, so how is Julien wrong? This becomes clear from a look at the principle behind signal generation and measurement principle:
F(c) = p*N*E^c
is the signal (fluorescence) measured at cycle c where p=proportionality factor, N=initial concentration/amount of amplicon, E=amplification efficiency (1>E
Hello Jochen thank you for your detailed response. I knew that the fluorescence was length dependent but did not know it was also sequence dependent. If we have the same amplicon sizes for different genes how much can the proportionality factor vary? And how could this be determined?
You can generate PCR product, possibly with outer primers, purify it, quantify it as precisely as possible (e.g. by OD260 or some fluorimeter) and use the same amounts from this purified (and diluted) PCR products as template in a qPCR to see how different the cT values are.
CAREFUL! Take care of contaminations! Perform the PCRs in a different lab, also the whole quantification! Only take the diluted aliquots back to the place where the qPCRs are pipetted.
Thank you Jochen to rectify my answer. I reply a little bit too fast to this answer, and I agree with you. Indeed, in my previous work, I compared the relative gene copy number of the same gene in different ploidy level state and not the the copy number of different genes.
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