We recommend to include – depending on the qPCR assay formate – the following controls:
No RT – control. If you work with a reverse transcript from mRNA as template, you should include samples without enzyme as a noRT control.
No Template Control (NTC) – Run each gene with NTCs using water instead of template to check for cross contamination.
Reference genes (“housekeeping genes”). For each sample several reference genes should be included to enable a subsequent quantification. Please check upfront that the reference genes are not influenced by the intended treatment of your sample.
Alternative Positive Control – For absolute quantification assays, in which no reference genes are used, an alternative positive control is required. This can include a control with a well defined amount of cDNA, that will show already known results.