How many replicate reactions should I run of the endogenous controls to demonstrate that they are equally expressed across my reference and test samples?
In general, the following experimental set up is recommended for each biological replicate: 2 RT reactions and 2 PCR reactions for each RT. It may not be necessary to test all the samples in your study, but at least one representative from each type or group of sample should be tested. It is important to realize that using as many controls as possible maximizes the chances of an optimal normalization procedure.
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