You got a bad reference there. The confuse the meaning of "housekeeping gene", "positive control" and "calibrator".

If you want to determine the CN of a target gene, you need to measure one other sequence of a constant CN as well. This other gene may be called a reference or a calibrator.

In contrast, the term "housekeeping gene" denotes a gene that is expressed (transcribed to mRNA) in any cell(type) at nay developmental stange, and at any time. Such genes were helpful for RT-PCRs -where mRNAs are reverse transcribed into cDNA which is then amplified in the PCR- as they could be used as general positive controls. In quantitative real-time RT-PCR, often housekeeping genes are used as reference genes, what can be ok but what can also be desastrous. A valid reference gene for quantitative RT-PCR needs certainly to be expressed, but only in teh cells being analyzed and additionally is needs to be expressed at constant levels in all experimental conditions you are analyzing. But this is relevant only for RT-PCR, not for CNA, where you amplify genomic sequences.

So back to CNA: From one sample you measure the Ct of your target gene and also the Ct of a reference gene (or reference sequence; some genomic sequence with a constant CN per genome; no need that this is a gene that could be transcribed). The difference in Ct values (ΔCt) is then comparable between different samples. You can analyze the mean difference in ΔCt values between your experimental conditions (ΔΔCt) to see if there is a systematic difference in the CN of the target gene. This would be all that is required for a relative analysis.

If you want actual CNs, you would need to select a reference with a known CN and you would need external standards to preform an absolute quantification of the target and the reference gene.

Dear Prof. Jochen Wilhelm

Thank you kindly for an in-depth answer. However, I still need some clarifications which I will appreciate if we could have a brief google or zoom meeting.

Thank you dearly

My understanding of the paragraph you cite is that the two are different things. The positive control is to ensure that your primers and conditions are working properly for your gene of insert. Hence you want to spike your sample with some cDNA of your gene which you know should amplify just to be sure that everything is working correctly and reproducibly for your target gene.

The "housekeeping" gene is much as Jochen Wilhelm described, you need some gene with known and constant levels of expression (at least in your tissue) in order to compare your unknown to. You might have different amounts of template in different samples, so you standardize against a known constant gene in order to do valid comparisons between different samples.

Thank you Michael J. Benedik