Dear Alifia,

Sometimes, you have to run samples in two different plates, here the calibrator will be a line or two line of the reference gene from the first plate to normalize any stochastic resulted by the machine or other effector.

In our lab we were using a machine that has a program to analyse the results and it calls the control sample as calibrator.

The value of the ref gene has to be considered from all samples to normalize the results. In other words, each sample has to be normalized first to its own ref gene. In an ideal RT-qPCR reaction the difference in the value of a ref gene for different samples has to be not more that 1.

Pls refer to the following link which contains a relevant discussion:

https://www.researchgate.net/post/What_is_the_calibrator_in_qPCR

Hello Alifia Rahma

I agree with Mohamed Khashan . The calibrator is used to correct for technical variances between different runs. You will have to normalize each sample with its reference gene (GAPDH) of the treated group.

The untreated group may be used as a baseline relative to the expression/detection level of target gene in the treated group. The relative quantity of a target gene in a treated group could be expressed as the fold change relative to an untreated group, using GAPDH, a reference gene, as a normalizer.

You may understand the calculations much better if you refer to the link below. The link below presents data from an experiment where the expression levels of a target gene(c- myc) and an endogenous control (GAPDH) are evaluated. The levels of these amplicons in a series of drug-treated samples are compared to an untreated sample.

https://www.science.smith.edu/cmbs/wp-content/uploads/sites/36/2015/09/Analyzing-your-QRT-for-relative-2%5E-%E2%88%86%E2%88%86Ct.pdf

Best.