I have a problem thinking on a paper discussion. When sb extracts RNA from a pool of embryos of different stages to analyze pattern expression of one gene: the number of cells impact in the quantity of this gene!? I mean, if there are differences in relative expression of the gene it could be due to differences in the number of cells, and not because of one cell have actually difference RNA content of this gene!? or it is not important because we work with exaclty the same RNA content to make c-DNA? I mean for example 1ug!
This is probably where your internal gene for normalization comes in. Ideally we will choose a gene that has an expression which changes linearly with the total amount of RNA (in this case, due to embryo growth). In zebrafish, EF1a is a good internal control. However, to be safe, I would carry out the RT using more than one internal control genes (eg. actin, GAPDH, EF1a) and calculate the geometric mean from values normalized against each of these controls. It's a little tedious but this would give you more robust data.
There will definitely be impact of cell number on the quantity of RNA. But at the same time you are using same quantity of RNA e.g., 1ug for cDNA synthesis. It seems you are analyzing some specific gene expression with relation to different stages of embryo development. Then in that case if you are observing changes in the level of expression with respect to the expression level of internal control gene/s e.g., GAPDH/actin, then your assay is giving you some indication of the changes.
A couple of additional comments: (1.) if only one cell type in the embryo makes the RNA in question, then your RT-PCR answer will tell you how much of the mRNA it maintains, but will also reflect the number of that type of cell in the embryo, which might change over time -- and probably does. (2.) RT-PCR results are very sensitive to degradation of the RNA prep, esp. if you prime with oligo-dT. Normalization does NOT address this, because the amplified region lies a different distance from the polyA tail in different genes, and of course nuclease sensitivity is somewhat sequence-specific. So, if you see any hint that your mRNA is degraded, don't use it.
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