Need more information to suggest anything.

What are the cytokines you are looking at and from which cells? How are you measuring the cytokines? You do not see cytokine levels in controls. Do you see any levels in infected or treated samples?  Are you sure that the cells you are using are known to secrete those cytokines. 

Thanks a lot @Rakeesh Ganji for your cooperation. 

I am using Raw 264.7 macrophage and lung epithelial A549 cells. My main interest is to see the iNOS along with pro-inflammatory cytokine IL-6 and TNF-a gene expression by RT-qPCR. I measured the NO level by using Griess reagent but  here also control showed higher NO level almost same as infected samples.

RNA levels may not always correlate with the protein levels. So, I suggest you to do ELISA for measuring cytokines and Western blot for iNOS. There could be several reasons for this discrepancy.

I encountered similar problems when doing such experiments and the reason was mycoplasmal contaminations. If your cells have mycoplasmal contamination even the control uninfected cells show higher levels of IL-6 and TNfa. I'm not sure of NO. Macrophages show secretion of pro-inflammatory cytokines with infection (could be any kind - here mycoplasmal contamination too). If that is the case, even your control cells show higher levels of these cytokines similar to your treatments. Make sure your cells are mycoplasmal contamination free. 

Hope this helps.

Hi Rakesh,

Thank you so much for your continuous support. It's always been a great pleasure to hear from you.  

Dear Hafij,

I see you got some good answers already. I wanted to point out that you may want to make sure your RT-PCR is working correctly as well. Specificity and precision are both very important, and this applies to target genes and housekeeping genes. If you tell me how you do your RT-PCR and what controls you had, I might be able to help more. For example, did you use SYBR Green or Taqman probes? How many repeats have you done for each condition? How close your primers were to the 3' prime end of the transcript? and how did you make your cDNA (oligo dT or random hexamers)?

Good luck with your experiments!