mike2k

Hi,

I´m new to this forum and I hope to find some help here.
I´ve recently started to isolate RNA from bacteria (spirochetes).

My RNA always looks kind of weird: Visualized in an "normal" agarose gel (but sample buffer containing formamide and formaldehyde) I see beautiful 16S and 23S bands. However, in the 5S region there is always a very big spot, which might correspond to more than one band. I have also run my samples through Agilents bioanalyzer - which can never generate a RIN - obviously because of this unexpected signal in the 5S region.

I assume this is degradation, isn´t it? What makes me think is that I currently use two different protocols (TRIZOL; Hot Phenol) - and the problem is with both methods. Another thing is that in the bioanalyzer if there was degradation I would expect a zick zack of the curve, which I don´t see. I have also to say, that I´m quite new to RNA isolation and that I´m not that experienced;  but I´ve taken every precaution that I can think of to avoid RNAse contamination (gloves, RNAZAP, DEPC, extra pipettes etc..). Maybe I have overlooked something but I cannot think of anything.

I´ve uploaded an example from the bioanalyzer. If anybody could help me in interpreting this result I´d be very grateful!

thank you!