Hello,

I am working with gut microbes that are quite difficult to lyse. By using a kit, it gives a very low yield. So I am trying out my protocol for total RNA extraction. The extracted RNA will be used for northern blotting. I have been pretty strict in maintaining an RNase free environment (using filtered tips, keeping cold, treating pipettes with RNase away, etc).. This is a 1% non-denaturing agarose gel though my sample has been denatured with formaldehyde buffer. The rightmost is DNA ladder (as a control) and the leftmost is yeast RNA 3ug as a control. Samples are loaded in 3ug. The 260/280 ratio is 2.2 and 260/230 is 2.3. I had to increase the UV exposure time quite high to see all these bands, otherwise only the two bands (23S and 16S) would appear. My concern here is that the 23/S/16S band ratio is flipped. Rather than it being 2:1, it almost looks like 1:2. Did my RNA degrade? What can I do to improve RNA quality? Thank you so much for your help!