What you are doing is called a "relative expression" experiment and is very common. Yes, you normalize to the expression of a housekeeping gene (e.g. if housekeeping gene is 1.0 is my gene X above or below that level?). You compare X to housekeeping and Y to housekeeping. That will also give you a way to compare X and Y to each other.

I'm curious why you are not including any sort of comparison to another cell line or is this one considered a "control/standard" in your research?

Hey Katie,

Thanks for your response! I'm familiar with traditional relative quantification methods comparing HKG and GOIs in control and treatment samples.

What I'm planning on doing here is slightly different, I've laser dissected neurons from a post mortem brain and I simply want to know if mRNA X or Y is higher within the population of cells I've collected (I do not need to know the absolute values). In theory, I don't see a reason why I cannot workout a ratio for the different cq values as it's the same population of cells with no differing conditions and I have primer efficiencies but I'm no expert in this field, I've seen other argue that this type of analysis shouldn't be done so I'm looking for clarity. I'm only interested in the relative mRNA jn these cells at present, in future experiments I'll compare to another cell type using standard relative quantification.

Thanks!

If you only want to compare gene X to gene Y to get a relative expression (X is higher than Y), then you can do this with qPCR, but I suggest you use multiple primer sets per gene to reduce bias due to your binding sites and possible splice product variation.

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