Davebrid: Wrote initial page

2013-12-02T14:57:17Z

Wrote initial page

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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.

Quantifying Relative Expression from qRT-PCR - Revision history

Davebrid: Wrote initial page