A standard curve tells you whether your PCR is well standardized, that is whether all the components used are in the right concentrations. Also, when you do not know the concentration of your template, you can run it with a serial dilution of known concentrations of your 'positive control'. Ideally, when you plot a standard curve of a serially diluted template, successive dilutions should be 3 Ct's apart. So. this way, you can extrapolate the concentration for your "unknown" tube. Also, what was the purpose of your comparison? I think the comparison will tell you which template your PCR picks with a better sensitivity/ LOD.
If I understand your question correctly, you should also run a standard curve on H5N1. Even if this standard curve is established using a dilution series from a stock of unknown PFU, the slope of this curve will allow you to calculate the reaction efficiency for the H5N1 M gene. If the reaction efficiency is identical for H5N1 and H5N9, then your comparison would be correct. If, for some unknown reason, that the amplification is much more or much less efficient for H5N1 than for H5N9, than the comparison cannot be made. It is very important to test the reaction efficiency for each strain.
Hi Ibrahim,
You should prepare individual standard curves for each gene of your interest. Standard curve is basically prepared for standardizing your PCR reaction i.e. the concentrations of primers and template you are using are enough good for your reactions. Also with the help of standard curve you can calculate your primer efficiency which is also an important factor for real time PCR reactions.
Thanks you all, so what I did understand is that standard curve is for validation of my experiment not for determination of quantity of RNA in my sample, or in other words (viral load, viral titer, viral nucleic acid quantity), if my understanding is right this mean that since M gene is quite stable in most of influenza strains, so the comparison may be correct, also no need to correlate the numbers to PFU, since I am not searching for viral load now,
Mark Laflamme, M gene is quite stable in most of influenza viruses, I am not comparing HA or NA which are quite variable,
Siddharth Anand, How can I known the concentration of my samples from the standard curve ? can you let me know, do you mean the value of CT ?, but I think this will be rough quantification and not absolute quantification.
hey Ibrahim,
to ascertain the conc. of your samples from the std curve you will need "standards" with known number of copies. They can be obtained commercially from sources like QCMD or you can clone the M gene, synthesize in vitro RNA and use the quantified in vitro RNA as your standard. PFUs will not give you the true LOD or PCR efficiency for your assay. But i think you should bother about that only if you are planning to make a commercial kit out of your assay. Otherwise the comparison between the two strains is good enough if the CTs are not drastically differing. If there is too much variation between the CTs for the two strains, you should check for variations in the primer and probe binding regions between the H5N9 and H5N1 strains.
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