Hi so I have developed an assay whereby I can detect certain mutational markers on qPCR and have developed a standard using synthetic DNA at around 300bp. I typically work in copy numbers so my standard curve goes from 10- 10^7 copies with Ct values at each one. Curve looks great, R2 close to 1
Now I have been working on some clinical samples which obviously have a mixed population of WT and MT. So I run two reactions one with WT primer and one with MT primer. My WT primer doesn't amplify MT primer so no cross reactivity happening. However the sample of DNA is genomic DNA so much larger in size not to mention the nature of it not being necessarily uniform in length. The original plan was to compare the Ct values to the curve but I don't think I can do this anymore. Genomic DNA copies are very different to copies of synthetic DNA. If I try and use concentration instead e.g. ng/ul it still doesnt work as 1ng/ul of genomic DNA is incredibly different to 1 ng/ul of synthetic DNA. So I am confused how is someone meant to quantify samples in qPCR when standards can often be much shorter DNA templates.
Any advice on this issue or some extra scientific theory that I might be missing would be greatly appreciated.
I agree that great dilutions are needed with genomic dna having 1 copy every 3,000,000,000 bases and pcr product being 1 copy every 300 bases but you could consider cloning the pcr product then having 1 copy every 5000 bases (or the length of the plasmid and product) and maybe having some inert nucleic acid added to minimise absorption on to the plastic of the storage and reaction plasticware
Hi Paul, cant quite do that. For starters the chemistry of the assay doesn't perform comparatively. For instance at 1000 copies on my standard ct value is around 22 mins whereas with one of my samples I'm getting 22 mins for about 5000 copies. Similarly at 500 copies with synthetic it is around 35 mins but I am getting about 28 mins with the sample. The assay itself performs differently with the samples than it does the synthetics. Also I'm actually not using PCR but rather LAMP, so the LAMP product is somewhat unusable. Not to mention my samples that have a low mutant frequency, diluting them too much is almost not an option.
Is it common practice in qPCR generally speaking to making standards the same size as the samples you will test later on? Surely this is an issue many people encounter with qpcr standards and sample testing.
Unfortunately I am not familiar with lamp but in general it may be possible to compare 2 sequences (normal and mutant) with a fixed standard curve even if the standard does not behave exactly like the samples so long as it behaves in a reproducible manner, It was the standard samples that I was suggesting needed diluting not the unknowns. Might it be that samples behaving oddly are less pure or have enzyme inhibitors present in the sample?
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