Dear Talha,

You should check your cDNA quality. Make sure that there is no degradation by loading them on 0.8% agarose gel. If you see smears, DNA is not good. If you see a band, even light, DNA is fine for PCR.

Also should check the amount of input DNA in each reaction by using nanodrop or qubit to check for DNA concentration. Too much input template can be inhibitory. Best Luck.

I definitely agree with Badreddine and would add to his answer that you should check the quality of your RNA before RT.

I see some primer dimers at the bottom of your DNA gel which is not ideal. I would increase annealing temperature a bit to get rid of primer dimers. Secondly, it could be better also thinking to design a couple of more primers to avoid primer dimers.

Thirdly (for cloning purposes), sometimes even you get a little band amplification, cut this band, purify and re-amplify it with same primers , it could work.

I think you must have a typo in your question. You cannot amplify a 480,000 base pair product with PCR. I assume you mean your expected PCR product size is 480 base pairs. You have very faint bands in all but two of your reactions. Try making new dilutions of your primers and check your template concentration.

Dear Talha,

You first extract a high concentration RNA and immediately make its cDNA and repeat PCR again hope so you will get your desire band.