I am performing qRT-PCR for zebrafish different organs and tissues using sybergreen and elongation factor 1-alpha elfa as a reference control. However, there is variation in the expression levels of my reference gene. Can the variation in expression levels after normalization be accounted as a real variation in the Ct values of the target gene?
Yes. If the Ct of the reference gene did not vary, then you would not need a reference gene. Normalizing to the reference gene usually leaves you with less unexplained variance than normalizing to any other known metric. That's why we do it.
It is normal for reference gene expression to vary across cell types. There is no gene that will be expressed at exactly the same level in all cell types. Reference genes are chosen because they vary less than other genes, so they are the best option available for normalizing.
For example, you can see that common reference genes such as GAPDH or ACTB show quite a lot of variation across human cell lines in this paper (doi: 10.1038/s41598-021-98869-x.): Article Identification of new reference genes with stable expression...
You could try searching in the literature for zebrafish genes that show less variation across tissues than elfa, but if elfa is the best option then you don't really have any other choice.
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