It is acceptable to perform qPCR without ROX. However, you said when you add ROX, the result is not that good (you mean so, right?). I think you should re-calibrate the machine to make sure every filter, light path, etc are work well, especially the ROX channel.

And I suggest adding ROX into the master mix instead of adding ROX each time you prepare the qPCR, so that the result would be more stable.

Hope this will help.

I think the result should be valid......

Hi Rajesh....

ROX is not always an optional component in qPCR ....there is some basis why ROX should be used....and this is also decided by the Machine being used....Certain qPCR machines recommend use of ROX so we should use the dye in those machines.....While others have some internal calibration and ROX is optional there.....

So we on our own should not decide its use .....In my opinion go through the manual of your machine an use accordingly...

ROX is used as an internal control or you can say passive reference dye to detect the certain amount of fluorescence even in non-PCR reactions. Re calibrate your filters in reference to ROX dye and put the reaction.

Thank you guys for your kind answer. Finally, I found the answer. As you have said, its all correct. So, finally, I got my optimized reaction and I am using Agilent Technology REAL-TIME PCR machine (Mx Pro 3005) and it works well now and my reaction is stable. I am getting good efficiency of range between 100 to 115 which is perfect. For this machine the acceptable efficiency range is 85% to 115%. So, I think the problem has been settled. Thank you so much for your unprecedented replies and helps. Thank you so much.