Slight overloading causes "u-shaped" bands, it is the middle section that runs faster actually, rather than the edges running slower. If you load half the amount of ladder, you can still see the bands and I bet they will be straight (and running a little slower, at approximately the position of your current edges of the bands).

Overloading In DNA agarose gels accelerates the running of the DNA because the ionic strength of the DNA changes the conductivity. That's why the edges run as they should be running, and the centre of the band is too fast.

What is the running voltage? It seems  your running voltage is high. good running voltage is about 80-100v .

Best regards

Hi Christine, your RNA don't seem degraded to me, the cDNA amount are so low that is impossible to answer the question, for this reason usually nobody run cDNA after RT.

Thank you, Jürgen for the explanation.  I'll try loading lower amount.  I used 5 uL in this case, as it was advised in the product literature.  

Thank you for the suggestion Mohsen, I used 80V in this case for 45 minutes.  

Thank you for the comment Valeria.  So nobody usually checks for cDNA quality anymore?  Do you still bother to determine your cDNA concentration using Nanodrop? Thank you.

Hi Christine, as you can see it is not useful to check the cDNA amount on gel or the concentration with nanodrop. It is more important to start from a good RNA sample, run on gel (as you did) and check the quality with nanodrop, perform DNAse tratment if you want to perform real time, for example.

In general you are sure that with good RT kit, adding the correct amount of your sample, your RNA was retrotranscripted at high efficiency. Next you have to add the suggested cDNA for your PCR reaction; almost all the commercial RT and PCR kits are good.

I have another tip for you..  That U shaped band is caused by diffusion into the gel after you apply your sample into the well.  Therefore if you load that lane last and turn the power on quickly you will reduce the diffusion and reduce the U shape..

Thank you Valeria for the helpful tips! :) Thank you also Daniel (I'll try doing that next time) and Azzreena.