To generate the standard curve, we first need to prepare four 3fold serials dilutions of cDNA template known to express the gene of interest in high abundance, as a calibrator sample. If you do not have a cDNA template that has high expression of the gene of interest, you can generate the calibrator by pooling the cDNA from your unknown sample. Next, setup PCR reaction for each serial dilution. Enter the arbitrary starting quantity as 27, 9, 3 and 1, or other starting quantity based on the dilution factor. Finally, run the PCR and determine their threshold cycle values.