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.