I tried to make the probe several times but it failed again and again. It usually turns out that the probe after hydrolysis is very very short (maybe around 50bp). I did not check the RNA before hydrolysis except last time. However, I saw a band (though very weak) last time, but it still produce very very short probe after hydrolysis.I use Sp6 polymerase to synthesize RNA, and the original length is 1.5kb. My ideal length after hydrolysis is 500bp.
I'm guessing maybe it's the phenol contamination of the phenol:chloroform extraction step because my 260/280 value is low (around 1.6-1.7), and my 260/230 is also low, which is like 1.6-1.8. Also the peak is not at 260, it's almost at 270.