What positive controls are typically included in qPCR and/or qRT-PCR experiments?
It is critical to include appropriate positive controls in a qPCR experiment to determine if false negatives are being detected in the experiment. Positive controls fall into one of 2 classes.
1. Exogenous positive controls refer to the use of external DNA or RNA carrying a target of interest. If these positive controls are assayed in separate wells/tubes from the experimental sample, they serve as a control to determine whether or not the reverse transcription and/or PCR reaction conditions are optimal. Additionally, exogenous DNA or RNA positive controls may be spiked into the experimental sample(s), and assayed in parallel or in a multiplex format with, the target of interest. These control reactions assess whether the samples contain any components that inhibit reverse transcription and/or PCR.
2. Endogenous positive controls refer to the use of a native target that is present in the experimental sample(s) of interest, but is different from the target under study. These types of controls are often referred to as normalizers, and are typically used to correct for quantity and quality differences between samples.
Within the RT2 Profiler PCR Arrays, the Positive PCR Control (PPC) wells contain a plasmid with a primer assay that detects a sequence it produces. This allows for quick confirmation of the performance of the PCR steps.
The RTC wells include assays that detect the artificial RNA that is spiked in to each sample during the cDNA synthesis step. This ensures the Reverse Transcription step proceeded as needed.