Hello Zeynep ZÇ Çeli̇k,

I suggest you re-run the experiment because an 8-cycle difference is huge.This would be unusual for a housekeeping gene like GAPDH, which should be relatively stable.

There might have been an error in the RNA extraction process for one of the samples.

The amount of total RNA loaded for each sample might have been different. The number of cells used to prepare the samples from spheroids and 2D culture could have been very different, leading to different total RNA yields. A lower Ct value means more starting material, while a higher Ct value means less starting material.

You need to re-quantify RNA. Re-check the RNA extraction and qRT-PCR protocols to ensure they were followed correctly for both 2D and spheroid samples.

Best,

The starting amount of RNA in both is 1000 ng. There is no difference in the amount of RNA.