Real time PCR standard curve

As blunt as possible: when performing real time PCR it is a routine step to run one PCR in order to plot a "standard curve" with several decreasing dilution ratios from your sample. what is the real purpose of this? how should results be used/interpreted?

Answer

You can use 3 or 4 dilutions- 1:1, 1:5, 1:25, 1:125

Purpose of doing this is to calculate the primer efficiency. Ideally primer efficiency should be 2 i.e. two molecules of DNA are formed in a round of PCR. So after n-rounds of PCR there should be $2^n$ DNA. However, this may not be the case always. You calculate primer efficiency like this:

• Plot $Ct$ vs $log_2(Conc)$


• Get the trendline (in excel) or use a linear regression command for other applications


• Get the slope ($s$) of the trendline


• Efficiency in this case would be $2^{-s}$.

Some people use base of 10 in the log instead of 2 (for which you have to do $10^{-s}$ instead.

This, you can directly use to find out how many copies are produced after n cycles i.e instead of $2^n$ it will be $x^n$ where $x$ is the calculated efficiency. This is particularly useful when you are calculating the fold changes using the comparative Ct method. For details see this article.