Failure to determine a CT value can be for a number of reasons - but it's more-often very low expression or a poorly designed qPCR reaction (e.g. bad primers, bat TM, etc.). If you think have a good reason to think the failure to determine a CT is because of too much sample, you could always dilute it. A dilution series of a positive sample is useful for determining the linearity of qPCR reactions in any case.

Do you mean the sample amplified very late (low concentration)? What do your controls look like?

What is your positive control? Does it amplify? Do you have a standard curve to confirm sensitivity and linearity?

You have first to make sure your PCR reaction is working. Once optimized, no amplification can be due to very low expression, inhibitors in your extracts or simply too much sample.

@ Katie A Burnette I performed the reaction for 8 primers in triplicates. All other primers except two have been amplified. and in control (3 replicas), the Ct value was 26.

Brian Bower Thank you for your valuable comments. From your comment,

(If you think have a good reason to think the failure to determine a CT is because of too much sample, you could always dilute it) Could you please comment on in which case we will go for Dilutions, and when we have to increase/ decrease the sample quantity.

Nezar Al-hebshi I have done the reaction with 8 primers except two all have been amplified including an internal control (gpd-3). So, I am sure that the reaction is working. But i have got Ct value in One of the reactions in triplicates for this gene and it was around 32-36 in each of the test sample.

so i calculated the fold change from that and it was around 32 fold increased.

So, I suspect whether is is due to high concentration of the test gene in the sample.

Thanks for your valuable comments and appreciate your suggestions if any.........

Sounds like it's a very low abundance transcript. Try increasing the amount of template in those reactions for that one pair of primers.

Katie A Burnette Thank you for your valuable suggestion. Got the gene, amplified.