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?
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2 Answers
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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.
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$\begingroup$ usually you would find a $log(conc)$ vs $Ct$ graph in examples for which the primer efficiency calculation becomes 2^(-1/s). Means the same but what I described looks neater to me :P $\endgroup$– WYSIWYGCommented Mar 24, 2014 at 14:00
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$\begingroup$ and why is normalization with housekeeping gene performed? $\endgroup$– KatzCommented Mar 24, 2014 at 14:20
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1$\begingroup$ Because these are supposed to be stable in their expression. That way they can be used to normalize for differences between the cells in the experiment. $\endgroup$– Chris ♦Commented Mar 24, 2014 at 14:40
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$\begingroup$ and may I normalize with housekeeping gene results of an "A" qPCR run and a interest gene results of another "B" qPCR run? (same experimental conditions) $\endgroup$– KatzCommented Mar 24, 2014 at 16:10
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$\begingroup$ it is not necessary that you should normalize with a housekeeping gene. The reference gene should be such that it doesn't get affected by the treatment. Usually housekeeping genes are not affected unless there is some global perturbation- in such cases you should use a spike in. You should use the same reference for replicates otherwise comparison is difficult- qPCR is not absolute quantification; it's relative. $\endgroup$– WYSIWYGCommented Mar 25, 2014 at 4:10
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Because the purpose of the standard curve (which actually results in a standard line) needs to be linear. If it is not linear (because of not enough values which are not linear distributed), then you quantification will be wrong. Ideally the line should look like this:
This picture is taken from this website, which gives a nice introduction into the topic.
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$\begingroup$ and why is normalization with housekeeping gene performed? $\endgroup$– KatzCommented Mar 24, 2014 at 14:19