What is it & how does it work?
Inter-run calibration is a calculation procedure to detect and remove inter-run variation. The experimenter measures one or more identical samples in different runs. These identical samples are called inter-run calibrators (IRCs) and they are used to detect and correct inter-run variation.
Detailed information is available in Hellemans et al., Genome Biology, 2007.
qbase+ allows inter-run calibration using more than one IRCs making it more accurate. It performs inter-run calibration after normalization allowing the experimenter to re-synthesize cDNA from the inter-run calibrator RNA samples.
It propagates the error introduced during the inter-run calibration procedure.
To avoid interpretation of inter-run calibrators as technical replicates, they should have a different sample name in the different runs (e.g. IRC1_a, IRC1_b, ...). Users should then indicate that a number of sample names actually refer to the same biological sample that is used as an inter-run calibrator (e.g. both IRC2_a and IRC2_b refer to the second inter-run calibrator, sample IRC2).
This procedure is known as setting the inter-run calibrators.
When do I need it?
Two different experimental set-ups can be followed in a qPCR relative quantification experiment:
1. Sample maximization method
- As many samples as possible are analyzed in the same run. This means that different targets should be analyzed in different runs if not enough free wells are available to analyze them all in the same run.
- Preferred method because the experimenter is usually interested in comparing the expression level of a particular gene between different samples. The sample maximization method does not suffer from (often underestimated) technical (run-to-run) variation between the samples.
2. Gene maximization set-up
- Analyzes multiple targets in the same run, and spreads samples across runs if required.
- Often used in commercial kits or in prospective studies.
Whatever set-up is used, inter-run calibration is required to correct for possible run-to-run variation whenever a target is screened for different samples across multiple runs