interpreting RNA agarose gel results? - (Jul/25/2013 )
I just performed a denaturing agarose gel electrophoresis to check the integrity of total RNA that I isolated from banked tissues using Trizol. Having never done this technique before I'm not entirely sure how to interpret the results. I have 2 distinct bands corresponding to 28S and 18S rRNA with no significant smearing. But the bands are undeniably the same intensity, not the 2:1 ratio that all the protocols say you should see. The bands aren't super bright, but I didn't use much RNA as my samples are very limited (~1ug per lane). See attached image. So does the lack of the 2:1 ratio mean my RNA is crap? Would using more RNA in the gel and cleaning the box with RNase Away (which I didn't do) potentially help my cause??
Any insight is greatly appreciated!
Yes, your 18S is definitely stronger than the 28S. You can quantify this with ImageJ quite easily (I've done a "Plot Profile" so you can see the relative intensities of your 2 bands (see attached). The Agilent Bioanalyzer explication of their RNA integrity number (RIN) will probably be interesting for you: http://www.chem.agil...5989-1165EN.pdf