Hello, every one
what is the mean of amplification factor,and other values that present with the standard curve result?
Does the higher PCR efficiency has a great indication?
see below attached photo
thanks
Zeinab
The slope of qRT-PCR should be between -3.6 to -3.1 but if slope is -3.32 then your result is perfect. Again PCR efficiency should be between 90-110% but best is 100%.
Hello Zeinab,
For 100% efficient curve your
correlation coefficient should be 1(ideal), but 0.99 is good
Slope shoul be -3.32(ideal), so it should come around it
The slope of your standard curve should be as close as possible of -3.32 (-1/log(2)), meaning that for each PCR cycle, you perfectly doubled your DNA quantity. It indicates the same thing than PCR efficiency: if your efficiency is 90%, then your slope will be -1/log1.9 = -3.58, if efficiency is 110%, then the slope is -3.1. Efficiency measures how well your PCR conditions (taq polymerase, primers, salts...) are able to replicate your DNA fragments. You should also be careful with the correlation coefficient, which is calculated with your standard points replicates, and measures the "strength" of your standard curve. It should really be above .99, and if possible above .995. Maybe you can try another couples of primers or different qPCR conditions, but I would not recommend to use your current settings. Good luck!
The Standard curve slope should fall between -3.3 to -3.8. I agree with the above answers that the slope should be close to -3.322 which means that the PCR efficiency is 1 or 100%. A slope greater than -3.322 which is what you have indicates a PCR efficiency greater than 100%. This may occur when values are in the non-linear phase of the reaction or can indicate the presence of inhibitors in the reaction.
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