Hi,

I will try to offer some insight, though I am not an expert.

If I'm not mistaken, "undetermined" Cq value(or CT as you call it) means that in all the cycles of your reaction the signal from the target DNA did not pass the threshold level. Meaning that you didn't have enough target DNA present. The same sample can have different results for two genes because of different reasons. One gene may simply be much more expressed than the other. You are doing Reverse Transcription, which is very sensitive to the RNA quality. If RNA is becoming degraded in your sample, depending on the gene sequence, sequence length and the primer position some genes may still work in the PCR reaction while others won't.

I'd advise to start by checking your RNA quality to make sure it is in good condition. I would check the primers as well to make sure they are the optimal for the genes and maybe increase the template concentration.

Hi Marusa,

Thank you for your answer. But I am using DNA here not RNA. This is real time PCR not reverse transcrition. My sample has enough target DNA also, the samples was positive in conventional PCR. The primers also in good condition because the primers are working good for other samples. I am confusing and can't detect any problem here.

Oh, I'm sorry, I thought you were using RNA because in your question you wrote qRT-PCR which stands for Quantitative Reverse Transcription PCR. If you mean just real time PCR it's called simply qPCR. Sorry for the confusion.

Still, if you are getting undetermined Cq values and everything else about the reaction is fine, I'd suspect the problem with the template, or maybe some other reason that the gene is not present in that specific sample?

But that specific sample is positive in conventional PCR.

So it was positive for both genes in conventional PCR and only for one gene in qPCR. Did you do duplicates/triplicates and all of them were negative/undetermined? Have you checked that there isn't a problem with that position on the plate in your qPCR machine?

I did duplicate, total 4 duplicate. 3 were ok. Only one is undetermined. Every time not in same position. I did 3 time.  Every time one sample undetermined but other duplicates of that samples are ok, and this occur in different different position each time. 

I also have made gene expression by qpcr using beta actin.. and my sample results were 34 and 36 in comparison of Beta actin which was 28..and I have three samples undetermined..