What is your reference? A gene on the same plasmid or on the host cell chromosome?

ARHGDIA gene, its from the host cell and not from the over expressing plasmid.

First question: have you run this on a gel to confirm it's a product of the right size?

My guess here is it's simply plasmid contamination. Assuming your expression cassette doesn't have a massive intron (and I can't think of any good reason why you would put one in), then your plasmid is just as valid a target for amplification as your cDNA prepared from RNA generated by that plasmid.

When you prepare plasmids, you generally prepare bulk amounts, and it doesn't take many molecules to contaminate a stock (via contact, aerosol, whatever).

And of course if you're routinely PCRing this region, you also run chances of contaminating everything with the amplicon as well.

Cq values of 32 correspond to ~10 template molecules in your well, so yeah: it doesn't take much to get a signal. Quite possibly you've just coated everything in your lab in a fine veneer of contaminating plasmid/amplicon.

The important consideration here is: what are your Cq values like in the actual +RT, treated samples?

The beauty of qPCR is it is quantitative: if your actual samples have Cq values of 22, and your NT control gives 32, just ignore it. That's 10 cycles, or about a thousand-fold difference in template concentration: in any other assay you would absolutely ignore such signal as background noise, and indeed in most other assays you wouldn't even detect such a signal. It's only because PCR is so sensitive that you're aware of this contamination anyway.

If your Cq values for treated samples are 28-30, then yes, the contamination is problematic, but a bigger problem with this would be that THIS IS VERY LOW EXPRESSION, and so probably your overexpression cassette isn't working.

It seems that you have a plasmid contamination in your lab :(

If the contamination is not so big that it interferes with yor quantification, then you can simply ignore it. The Cts of the positive samples should be at least 3 cycles lower than the Cts of the NTCs. Otherwise, you are really in trouble. That's hard to eliminate. Ideally burn down the lab and re-start... or go to a different lab (ideally in a different building), or start over with a new plasmid.

EDIT: I just read what John wrote. It's essentially what I wrote, too (except for my sarcastic advice to burn down the lab in order to effectively eliminate a plasmid contamination).

Thanks for your wonderful brain storming. Answers for your queries.

1. Ran the plasmid on the gel and also verified by sequencing.

2. ct values are

Cells with control plasmid ct is 26

Cells with plasmid containing gene of interest ct is 8.5 (also this is very low, can this be acceptable. Same value with two diffetent set of primer)

Because of the ntc and rt negative, can i trust the above values.

You can/should dilute your samples by a factor of about 1000. This will give ct-values for the positive samples of about 19 and (hopefully) also shift the cts of the controls to higher values. Very small cts can be problematic, because the software has too little data to estimate the background. As a rule of thumb, ct values should therefore be larger than 15 (this is not always critical - but it can cause problems that are easily avoidable).

Thank you for ur ideas , I have tried the 1000 fold dilution. Now the control sample ct is around 28 and the over expressing plasmid samples are around 20. In NTC no amplification. I got around 95 times fold change. So, if I want to calculate actual fold increase upon over expression, should I multiply 95*1000 ?

As all samples were diluted with the same factor, this cancels out when calculating a fold-change.