I have nhe6 KO mice, confirmed via genotyping from their tail clips. I isolated BAT, iWAT, and gWAT from three controls and three KO mice. RNA isolation followed by cDNA preparation was performed from these isolated tissues. On running qPCR, I expected low ct values (high nhe6 expression) in controls and high ct values or undetermined ct values (may be?) for KO samples. However, shockingly! I got similar ct values (30-32) for both- controls and KO.
The questions here is-
Is it correct to perform qPCR on genomic knockouts? AND WHY?
30-32 is a bit high for Ct values, especially if you expect Nhe6 to be highly expressed in those tissues. What was the Ct value for your water control? The most likely answer is that the qPCR primers are poor. When doing qPCR on a "new" gene, I always test at least two qPCR primer pairs to ensure I get the same result.
The other possible explanation for a high Ct is just that you didn't include enough cDNA in the reaction. Did you measure a housekeeping gene, and were those Ct values more reasonable?
Alternatively, you might've included _too much_ cDNA in the reaction (cDNA synthesis buffer is inhibitory to PCR, so you want to dilute your cDNA after synthesis, ideally).
But like Ciaran Daly says, Cq values of 30-32 are far too high to be trustworthy: these correspond to countable numbers of target molecules (like, 5-20 targets per well) and are thus very susceptible to stochastic noise, mispriming and just general PCR shenanigans.
It would be helpful if you could provide more detail about your entire experimental set-up, since there are lots of places where things can go wrong.
How are you isolating the RNA?
What are your yields?
Are you QCing your RNA afterwards, and how?
What are your 260/280 values, and more importantly, your 260/230 ratios?
How much RNA are you using for cDNA synthesis?
How are you priming (oligodT, random priming, or both)?
Are you diluting your cDNA afterward?
How much cDNA are you using per well, assuming 1:1 conversion?
It might also be that the gene is not expressed (much) in the tissue and the knock-out is leaky.
Is the amplification specific? Have you checked that on a gel? What's the ct of the negative controls? What positive controls do you use?
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