Agarose gel for determining RNA quality (image attached) - (Mar/06/2015 )

Hi everyone,

I snap froze mouse adipose tissue, stored at -80C, and used TRIzol to extract RNA. I also treated the RNA with DNaseI, then re-precipitated and dissolved the pellet in TE buffer. I measured RNA concentration using the NanoDrop, and there was some loss of RNA after DNase treatment. I decided to check for RNA integrity by loading 1ug of RNA onto a 1% agarose gel with ethidium bromide (see file attached). I ran the gel for about 90 minutes and 90 Volts. I'm not too sure if my RNA is of good quality. In the first lane (from the left) is a DNA 1kb marker. The other lanes represent the RNA samples. I do see 3 bands representing the 28S, 18S, and 5S/tRNA, but I can't determine if any degradation has occurred. Some say that the 28S band doesn't necessarily have to be twice as intense as the 18S band. Any help would be appreciated. Thanks!

You should run a denaturing gel for rna with an rna standard.

You can improve your gel image by adding DNA dye to the positive buffer pool, which will eliminate the dark shadow at the right. You can also switch from using Xylene cyanole and bromphenol blue dyes to orange G in your loading buffer to eliminate the masking dye bands. I don't know what your ladder is, but it seems likely that your RNA is degraded, since the larger size band is dimmer than the smaller.

Using denaturing gel will help you to determine whether you have good quality RNA not. Again remember 28S has to be as twice intense as the 18S otherwise you should suspect some degradation.