I would like to compare gene expression in samples of leukemic mice. I found that Ct values are consistently lower in one type of leukemia than in the other. I have tried several housekeeping genes and other genes, and I always found the same tendencies for all of them. What could possibly be the cause of these differences? I performed the RT with the same amount of RNA (1000 ng).
And that is exactly why you use the housekeeping genes - so you can normalize & compare your data between your groups.
I bet once you analyze the data using the housekeeping controls, those differences will not be significant.
It's likely due to differences in starting RNA quantity and/or quality (which you can't measure by nano drop).
(1000 ng), the amount is quite high, try to reduce the amount and see.
One group might have poorer RNA quality or reduced cDNA than the other for whatever reason (collection, storage, less reverse transcription enzyme added, other pipetting issue during cDNA synthesis, PCR inhibitors present, etc).That's why we perform statistics on the delta Ct values (Ct of your gene minus Ct of the internal reference). It controls for those technical issues which are seen in every experiment. There may also be biological variables, e.g. cell growth differences. Try scrambling the order in which you process samples and addressing any technical variables. If the difference doesn't go away, it might be biological. It's usually not, but I have seen it before when working with RNA binding protein overexpression experiments.
I don't think 1000 ng RNA is too high. Check your cDNA RT recommendations per volume, but 1000 ng is usually within the recommended ranges I've seen.
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