Reference calibrator - Information
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In delta-Cq quantification the difference between two samples (true unknown and reference sample) is transformed into relative quantity.
Any sample can be chosen as reference calibrator (e.g.: real untreated control, sample with highest expression, ...).
QPCR calculates the reference calibrator (don't confuse it with reference/housekeeping genes, or reference samples---used for calculating the final fold change)
by taking the arithmetic mean of all samples of a gene within one plate.
By using only the values within a plate inconsistencies between the plates are not considered in the early steps of the analysis (they could alter the final results). They will be taken into account
when calibrating the plates (where the same sample/target combination needs to be present on all plates).
By ticking the "average reference calibrator across plates" box the reference calibrator is calculated by taking the arithmetic mean of all samples of a gene over all plates.
This affects multi-plate analysis where samples are spread across several plates.
Please keep in mind that ticking this box is not preferred and should only be carefully selected.
Side Note: it has been introduced to guarantee backward compatibility.
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QPCR - Information