Sabahat Fatima

The presence of contamination mostly arises from the surfaces on which we operate. Ensure thorough sterilization of all items using DEPC and also subject everything to autoclaving. Consult your colleagues to determine whether they are encountering the same problem. Ensure that the instrument is properly maintained, as you are already using new sets of all components.

For the NTC use HPLC grade or nuclease-free water and check the primers with the other cDNA samples if again the problem persists problem with your fluorescent dye or other reagent.

All the best.

I'm not seeing any contamination. The amplification is so low that the scale on the Y-axis is just over-emphasizing any values that are basically 0.

Is U6 supposed to be a low-expression control? Might want to check your original RNA sample for degradation. How much RNA did you start with? Does anyone else in your lab use U6 with stem-loop primers? See what sort of numbers they get & the exact protocol they are using.

Good luck!