For two step reaction, many people use the same HKG data from the same cDNA. But if you have new reverse transcription (RT) reaction, even if it is from the same RNA tube, you will have to run HKG again since the efficiency of RT might differ from run to run. For one step reaction, If you have enough samples, it is best to run HKG and your GOI at the same time with the same reagents to minimize the variation between different plate preparation or machine condition.
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