Hi Vipul,

is it possible, that your PCR is not optimal working e.g. because of impurities in your samples? Thid might affect also your signals. You could try to dilute your samples before going into the RT step (in case your target gene Cq would allow this). Otherwise you might think of trying another chemistry to check this

In general I would propose to use more than one Housekeeper and use a calculation methodology that combines them like Livak& Schmittgen.

Sven

Variation of 10 cycles (18 to 28) is unlikely to be gene instability, especially with something fairly ubiquitous like eEF4. As Sven suggests, it points the finger much more firmly at either

A) poor quality RNA

B) poor cDNA synthesis

So as ever: more detail would be helpful. What sort of samples are these, how long post-mortem do you collect them, do you take multiple samples from the same animal, and what sample handling methods are you using?

How are you isolating RNA?

I am assuming you're quantifying your isolated RNA via nanodrop or similar, but are you running any samples on a gel (or using a bioanalyser) to test for RNA integrity? Are you making sure your 260/230 ratios are close to 2.0? (low 260/230 ratios are a common problem with guanidium isothiocyanate methods like TRIzol, and contaminating salts will inhibit cDNA synthesis very effectively)

Before we can even address your housekeeping issue, it seems prudent to establish your methodology is sound and that your samples are of good quality. Sorry to be a bore.

TRIzol was used. Samples are reproductive tract tissues, procured within 30 min of slaughter. RNA quality was assessed by both : the nanodrop as well as running on gel. Efficiencies for qPCR were also calculated. As I mentioned all MIQE guidelines were followed.

So all had 260/230 ratios of 2.0 or more? (or at the very least, 1.7 or more)?

This is the biggest culprit of poor subsequent performance in my experience. Do you have primers for 18S? If you see the same huge variation in your samples for 18S, that's a sign you're looking at RNA isolation/cDNA synthesis issues.