Hey, We are testing a qPCR instrument that requires fairly large reactions volumes. To do some comparative studies, we simply used smaller reactions volumes when running comparative samples on a traditional qPCR instrument. However, we seem to get quite high variation between the instruments when calculating copies/µl, but almost identical concentration when it is measured in copies/rxn. The two reaction types are 30µl reaction with 6 µl DNA and a 10 µl reaction with 2 µl DNA. The components in the two reactions are the same and in the same ratio see below. 30µl reaction 10 µl reaction MM 15µl 5µl H2O 3µl 1µl Assay 6µl 2µl DNA 6µl 2µl Total 30µl 10µl Our standards are composed of synthetic DNA, for 30µl reactions we use 6x10^0-6x10^5 stds and for 10µl reactions we use 2x10^0-2x10^5.
This is how I normally would calculate absolute quantities: 1: 10^(Ct value-Intercept)/slope = Copies / rxn Then to obtain Copies/µl 2: (Copies/10 µl rxn) / (2 µl DNA/ 10 µl rxn) or (Copies/30 µl rxn) / (6 µl DNA/ 30 µl rxn) which would yield Copies/2 µl DNA or Copies/6 µl DNA. 3: Hence to obtain our copies pr. µl we would divide with 2 or 6 µl depending on which reaction volume is used.
However, I feel like something is missing or at least not accounted for. My arguments are that there are equal amounts of DNA copies (or DNA µl) per volume reaction and hence there is no difference between the reactions due to the concentration within the reaction itself. qPCR is based on fluorescence which is determined by the concentration. It is therefore not directly the absolute number of DNA template which determines the quantity but rather the concentration within the reaction. 2µl DNA / 10µl reaction volume or 6 µl DNA / 30 µl reaction volume yield the same concentration of 0,2. However, if this is correct, how to I proceed from here to calculate standardized copies/µl for both volumes?
It looks like you don't know the concentration of your DNA because you only give their volumes (in ul) but not their masses (in mg.) or concentrations (in mg./ul). You need to standardize your DNA concentrations (as well as the concentrations of other reagents used) and your results will agree no matter what reaction volume you use (assuming you are doing the experiment right).
Hey both, thanks for your answers, let me elabroate a bit on this. My synthesized DNA used for the standards have been measured by a qubit fluorometer. Using the molecular weight of the amplicons and the measured concentration found on the qubit I could estimate the amount of templates in the stock solution. The stock solution (1X10^5 copies/µl) was then diluted to match the appropriate standard, so for the 30 µl reactions we used 6 µl of 1X10^5 copies/µl (6x10^0 - 6x10^5 stds) and for the 10µl reactions we used 2 µl of 1X10^5 copies/µl (2x10^0 - 2x10^5 stds). The samples have also been measured on qubit, but mainly to assess potential inhibition. I would not be able to assess my samples based on the DNA weight as they are environmental samples and contain DNA from many organisms and not just one.
However, my point is that the 10µl reaction and 30µl reactions are exactly the same it is simply just a scaling factor. Even the concentrations of the templates in the standard reactions are the same. Hence I cannot justify diving with two different DNA volumes as the reactions essentially are the same.
E.g. 200 copies / 10 µl reaction volume = 20 copies/ µl reaction volume
Or 600 copies / 30 µl reaction volume = 20 copies/ µl reaction volume
Hope it makes sense.
I do not really understand your calculation. Why do you want to account for the reaction volume? I would do it in the following way: (1) use a standard dilution of known copy numbers (copy numbers per reaction; 2 - 200000 or 6 - 600000 in your example) for generating the standard curve. (2) measure your unknown samples and calculate the copies per reaction using the standard curve. (3) adjust for varying volumes of the unknown samples used for PCR (copies/rxn divided by the volume of DNA => copies per µl DNA). Then your results should be similar - independent from the used qPCR machine. Are they?
Hey Norman,
Thank you for your reply, would you elabroate on which calculation?
I get almost identical copies/reaction for the two instruments. However, when I try to infer copies/µl there is obviously much more variation because the reactions are divided with the µl of DNA for which there is a threefold difference between the 10µl and 30µl reactions.
Put it in another light, what do we actually measure when we do qPCR? It's the fluorescence, which is determined by the concentration of the templates in our reaction and not the absolute quantity alone. If the concentration is the same we should get the same Ct and hence the same copies/reaction, correct? This would normally not be a problem if the reaction volumes are all the same as everything would be relative to each other and hence directly comparable.
Hey Brian,
I think you do not need to account for the reaction volume (but for the volume of sample you put into the qPCR). Moreover, the concentration of your template in the reaction is not important (at least for a large range of copy numbers). What we really measure in qPCR is the concentration of the PCR product (fluorescence) and if a certain, constant threshold is reached we can estimate (from the Ct value)the amount of template at the beginning of the PCR.
To your problem: if I understand you correctly, you put different amounts of template (2 or 6µl, for both standards and unknown samples) into the reactions in the different cyclers. If you calculate the copy numbers per reaction you get similar results (despite different amount of templates)- thus, if you calculate the copy numbers per µl template you get larger deviations. Is the PCR efficiency calculated from the slope of the standard curve equal between the cyclers? Maybe you can upload an Excel file with your data?
Hey Norman,
"What we really measure in qPCR is the concentration of the PCR product (fluorescence) and if a certain, constant threshold is reached we can estimate (from the Ct value) the amount of template at the beginning of the PCR." Well everything would be relative to the starting concentration of the reaction. Even the PCR product we measure the CT with should be affected relative to the starting concentration?
Samples are taken from the same source but their extraction and qPCR analysis (as described) differ. Extraction methods are different, but they are made to mimic each other and they have been verified by extracting same source of DNA and gives comparable results. The instrument (instrument 2) providing the lower copies/µl had a slightly higher DNA yield than the other method.
I would expect some natural variation between instruments since the samples are not completely identical. However, what is unexpected is that one instrument seems provide much lower estimates of copies/µl.
I've attached some of my data so you can get an idea of what it looks like. Graph illustrating the copies/µl is calculated as described above.
Hey Brian,
it is hard to find the real cause for the variation in your data without having all information. At least it seems to be no constant deviation (all samples) between the cyclers.
If I understand you correctly, than the samples are not the identical DNAs. I think this is potentially the most likely cause- can you run the identical DNAs on both cyclers?
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