I have encountered this problem as well. Despite using sufficient reference genes, you need to normalise for inter-run variation if you divide your target (primer pair) in different plates. What you can do is have a few (good) samples and run them on all plates (same primers) and normalise to those samples. I would suggest to take at least 3 different samples to have a good normalization. Even if you have already performed this qPCR analysis, you can still analyse a third plate, containing samples from both plate 1 and plate 2, and normalise those values according to the results of plate 3. If the conditions are optimal, same temperature, same mastermix, they should not differ that much. In reality, it could vary a little bit, and therefore you need this adjustment. I have been using qBase software.

https://support.biogazelle.com/hc/en-us/articles/212550205-Inter-run-calibration

If your data is not that big, you can perhaps even to this manually.

Good luck!

Hi Wardah,

If you are not already, use of a passive reference dye such as ROX can add an extra layer of confidence to your results.

ROX passive reference dye is an inert additive that provides a constant fluorescent signal for sample normalization.

The fluorescence emission intensity of the reporter dye is divided by the fluorescence intensity emission of ROX. This ratio is the normalized reporter intensity (Rn). This is then used to look at the reporter fluorescence above baseline to increase the accuracy of yoru results. Most qPCR software will calculate this for you.

Ultimately this is used to correct for differences in optical paths between wells, runs between plates and liquid handling errors.

Agree with @ Simon Strobbe

Regards