Amplification Effeciency - solve the problem with E value (Jun/18/2008 )
Hi everyone
How can you caculate the E value? by standard curve or by flourescence signal directly from amplification data. I use the second method to get the E value (by LinRegPCR software. Anyone know this program?)
For each reaction tube, I get one amplification curve and then from that I get one E value. But it is funny that I can not get the equivalent E values for the same gene, even they have big difference ( for example: 1.02 and 0.89 and so on). I also try to be careful when set up reaction such as: diluting, pipeting etc..
Anyone who know, please tell how I can solve this problem?
Thanks alot
How can you caculate the E value? by standard curve or by flourescence signal directly from amplification data. I use the second method to get the E value (by LinRegPCR software. Anyone know this program?)
For each reaction tube, I get one amplification curve and then from that I get one E value. But it is funny that I can not get the equivalent E values for the same gene, even they have big difference ( for example: 1.02 and 0.89 and so on). I also try to be careful when set up reaction such as: diluting, pipeting etc..
Anyone who know, please tell how I can solve this problem?
Thanks alot
I've always used the slope from the standard curve to calculate efficiency (E). The equation is E=10^(-1/x)-1, where X=slope of your standard curve. See this fig (http://www.biomedcentral.com/1471-2407/6/33/figure/F1?highres=y) for an example.
good luck
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I've always used the slope from the standard curve to calculate efficiency (E). The equation is E=10^(-1/x)-1, where X=slope of your standard curve. See this fig (http://www.biomedcentral.com/1471-2407/6/33/figure/F1?highres=y) for an example.
good luck
[/quote]
I know this method, but actually E value is affected by template concentration.
anyway, thanks for your reply