Inter-Run Calibration

You need to do inter-run calibration if you want to compare samples from different runs e.g.:

• when it is not possible to get all samples for the same gene on the same plate
• when you do additional runs weeks or months after your initial experiment

Of course there is a lot of variability between runs on a qPCR instrument:

• thermal block is not always heating uniformously
• quality of the lamp, the filters and the detector decreases over time
• data analysis settings on the qPCR instrument (baseline correction and threshold) can be slightly different
• efficiency of reagents (polymerase, fluorophores) is variable
• optical properties of the plastic plates vary

Fortunately, inter-run calibration allows you to eliminate most of this variability.

In this experiment we will analyze the data from the gene expression experiment (see Analyzing gene expression data in qbase+) together with data from 2 runs (Run4 and Run5) that were done weeks after the initial gene expression experiment.

Because the data comes from two different experiments spread over time, we have included three inter-run calibrators on the plates: Sample01, Sample02 and Sample03.

The principle of the IRCs is very similar to that of the reference genes: In theory, the IRCs should have the same NRQ in each run. In practice, the difference in NRQ between two runs is a measure of the inter-run variation and can be used to adjust the NRQs to remove the inter-run variation.