I would say that your CD133 primers did not work. Have you used them in other assays/cells and they worked? If yes, then it is a problem of this particular assay, probably the aliquot of the primers. Using a new aliquot of the primers should solve the problem. If it is the first time you use these primers, optimization may be necessary or the primer design did not result good. The cheapest solution would be to design new primers.

Did u check your primers before starting your real time PCR and annealing temeperature conditions I advise you to check your primers by doing a normal PCR If you are not getting a single band then there is a problem with the primers but if you are getting a single band you need to troubleshoot and set your reaction carefully again and even make sure that your RNA or cDNA is not degraded before use.Good Luck :)

Dear Konstantinos and Aneesha,

thanks for your answers... I used Taqman Primer for CD133 gene( Hs01009250_m1 ) that was bought from Life technologies. Taqman primers are optimized so there is no optimization problem:) . I couldn't get a signal from CD133+ subpopulation of cancer cells nut interestingly I got signal from CD133- subpopulation of cancer cells. I used Trizol reagent to isolate RNA from my subpopulations. Differences between RNA of subpopulation is that RNA of CD133- subpopulation is more pure than CD133+ subpopulation...

Dear Emre,

You may want to test these samples for another gene known to be more expressed in CD133 positive or negative cells to be sure there was not any sample swiching. 

If RNA quality is a concern, you could test a couple of genes with low expression to see if they work in both samples. 

Good luck