I have already assessed many possibilities with no positive outcome. While preparing the negative control, I usually avoid any contact with RNA, gDNA or any kind of template that could potentially contaminate the control.

I also wondered if it was my handling, but in the second round, the negative control turns out well.

Then I also changed all my reagents to new ones. I always thought it could be the water or the primers used. I have changed many times these primers, and what intrigues me most is that these primers are the ones we have always used and always have worked.

Also pondered the possibility if when I loaded the samples in agarose gel, perhaps one of the samples might have leaked/spilled over the well where the blank was loaded, but it wasn’t the case either.

My guess are the primers or even the RT enzyme. I would appreciate any further insight provided.