Looks like you already know what you have to do to find out if your mRNA expression is low or high. You need to use qPCR to compare to the expression of a "housekeeping gene". Or do an RNAseq run and compare to a housekeeping gene. That's the only way to compare your samples to each other.

Katie A Burnette Thanks for your answer, but it will always be a relative expression this way... I am confused in a situation that I am comparing my samples with already high expressing pool... The problem is i am not working with treatments (where relative decrease or increase makes sense), but different human samples... so although my samples have relative value of 0.7, I am sure its still high...

I have problem in deciding some cut-off value where I can recommend my sample as high or low in mRNA..

I really don't understand why you think this matters, nor why you're using a pool (?).

Are you pooling all your cDNAs and then comparing individual cDNAs to that pool? If so....why?

As far as gene expression goes, raw Cq values of less than 20 usually represent very abundant transcripts, 20-25 moderately abundant, and 25-30 low abundance, but this doesn't necessarily translate to protein levels, nor should "amount" be an absolute metric: take something like beta actin. This is a very abundant transcript (thousands per cell), and if you reduced it by a factor of five-fold, there would still be a lot of transcripts (abundant expression), but crucially this abundance would still be five times lower than cellular demand. Abundance per se is not a useful metric here, whereas relative gene expression is.

John Hildyard thansk for suggesting about Cq values. I am using a pool (of 3 cancer cell lines) to standardize different runs and compare different samples...

as far as why it matters, according to my sample i have relative values from 0.18 to 4...now 0.97 may seems low but its Cq value is still 19..so its actually high expressing mRNA..

Doesn't matter: it's all relative. If a high expressing mRNA is reduced by 5-fold, it will still be high expressing, but it will nevertheless be 5-fold lower than cellular demand.

If you reduced the amount of aluminium in a car by 80%, the car would still contain WAAAY more aluminium than gold, but it would nevertheless have nowhere near enough aluminium for normal function. It's the same with mRNAs. High-expression transcripts are high-expression for a reason: the cell needs loads of them, or they're really unstable. Reduce them by a lot and you'll still have loads, but not loads enough. Biological consequences are likely still significant.

Your question is not clear.

Sorry Guys for the delayed response, I was too busy..

John Hildyard thanks for your explanation its way clear now for me...

Andreas Eisenreich i am sorry, if the question is complicated, but here it is again:

I do qPCR using beta actin as a housekeeping gene and compare it with a pool of 3 cell lines using deltadeltact method..

I have different samples, the relative expression using delta delta CT for my samples ranged from 0.5 to 7... However, the Ct values were between 18 to 23...so although 0.5 does seem look lower but for me Ct value of 22 or 23 was still very high... that confused me why should i take it low as ct value is still abundant expression..and given John point, it should be considered low as it still might be high but not high enough...