Hi Aya Youssef

There are a bunch of causes can be considered.

1. you have to check if your cells or experiment samples do express TNF alpha.

2. If you did check that, you can add a positive control to eliminate some causes.

3. Check the amplification curve and melting curve.

4. How much was Ct of B-actin? maybe you used a low concentrations of the sample or the you did not adjust the reaction efficiency.

If you can give more details it would be easier to handle the issue.

All the best...

Hi,

3 microlitre cdna, this is too much.. I would suggest to check the Ct values for B-actin first. Check your primers too.