View source for Quantifying Relative Expression from qRT-PCR

Normally you will have several replicates for each primer/sample pair.  This should start off as 3-4 and only be reduced when you are confident that the extra replicate is not helpful

==Data Processing==
* Align the Cp values for the replicates for each sample/primer pair beside each other and compare.  If necessary remove any failed runs.  This should also tell you if there is a consistent bias in a particular channel.
* Average the Cp values for each primer/sample pair.  Arrange these such that the samples are grouped together.
* For each primer/sample pair '''subtract''' the control value for that sample.  This will give you the normalized Cp values.  See Selecting a Control Primer below
* Take 2^-Cp for the normalized Cp value.  This converts the Cp values into arbitrary normalized expression values.
* Calculate for each primer, the average for just the '''Control''' group.  This will be the control average
* Divide all the arbitrary normalized expression values by the control average.  This will set the control group to average = 1
* Calculate the mean, standard deviation and standard error for each primer set and for each group.  The control averages should always be equal to 1, or else something went wrong.

==Selecting Control Primer==
* For each sample and treatment set test several control primers.
* The goal is to find a control primer that does not change with the treatments.
* To test this, calculate Pearson's correlation coefficient for each potential control primer pair.  Find two primers that correlate closely together.  Chose one of these.