In my opinion, if everything is good, and the primers are verified, you don't need to perform a gel in the first place. Still if you want to do it, just increase the DNA loading on gel as mentioned above.

No, you shouldn't increase the cycles past 40. After 40 cycles, all you are doing is creating artifacts.

Why are you running a gel to look at your product concentration? The entire point of doing qPCR is to use the Ct values to calculate your product concentration. The melt-curve is also rather uninformative since you can have similar melt-curves for different products.

What you need to do is you your standard curve to check your primer efficiency and then calculate your product concentrations to know the expression of your miRNA of interest.

Good luck!

Thank you all for kind replies!

I have checked the primer efficiency in the first place before the main experiment. It turned out to be about 95% (cannot remember the exact number) according to the standard curve, so there should be no problem with the primers.

I agree with Katie; the gel image was not in the original plan, but I was asked to include it from a referee of my manuscript... Anyway, the best thing to try would be simply increasing the input. Thanks again!

You told in your answer above that you made a standard curve; how did you do when performing gel electrophoresis? Or do you apply qPCR (=real time PCR) as well?

If you apply qPCR it is simple to check if by-products appear when you go up to 50 cycles

I agree with Katie that even though the gel was asked by the referee, you can submit the melt curve analysis graph.

So you perform qPCR and you can produce melting curves.

If your primers are really OK, you should make a standard curve with your positive controls with a Cq varying from, say a Cq of 25 to 35? You can do that at 50 PCR cycles.

Near a Cq of 35 you are almost at a single copy in your PCR mixture.

If you melting curve is OK at all dilutions, showing a single product peak and no peak at all in the most diluted samples you can trust a high Cq.

I don't see why you want to make a gel electrophoresis; it's much less sensitive than a melting curve.

For further information, see https://www.springer.com/la/book/9789811316036

chapter 6 about tecnical sensitivit and specificity.

I'd say you could try loading a gel with a larger sample volume to demonstrate that the product sizes are as expected (if that's what the review would like to confirm). If the reviewer thinks that the gel image is somehow quantitative, then you can politely tell them that the Ct values show quantity much more accurately than an agarose gel. Your reviewer might not know much about qPCR.

To add to the answer of Katie, I wonder if the reviewer knows of the low sensitivity and specificity of gel electrophoresis if compared with qPCR. That differs log- scales. If performend adequately with optimised primer/target combination you can detect one copy of a target in your sample with qPCR at a Cq of about 35. That is absolutely impossible with agarose electrophoresis even if you load more sample. You can refer to Fig 5.11 and 6.29 of the book Molecular Diagnostics Part 1: Technical Backgrounds and Quality Aspects ISBN 978-981-13-1604-3

I also agree to the aforementioned comments.