2 replies to this topic

[vegeta]

Im trying RNA prep for QPCR using the qiagen RNeasy kit. Im pretty sure that I was really really careful while extracting RNA. I added beta-mercaptoethanol to the lysis buffer, homogenized the lysate and froze it at -70. I did the prep after a week and I was pretty careful while doing it, so I doubt I made any mistake there. I didn't have enough RNAse free water so I pretty much used old TAE made in our normal distilled water. Since I was really anxious to see how it went, I used the DNA loading dye and DNA ladder because we didn't have the RNA versions. Heated the samples at 60 C for 5 mins and ran the gel at 130V (the electrophoresis tank is quite big, so I don't think the V/cm wouldve gone too high).

The RNA was degraded with a smear at 1.5-0.5Kb. I just wanted to see what happened if I ran the gel for another 40 mins. Interestingly after 40 mins of running, the ladder was still there (just separated more) but the RNA lanes were empty!!! I couldn't see the smear I was seeing before despite the equivalent ladder being right in the middle. I think it ran off. But does this mean that RNA got degraded DURING electrophoresis (became progressively smaller with time and ran off)? Do I need to add DEPC to the TAE? I got some RNA zap to clean the tank. But, what do I do with TAE. Can distilled water have RNAse?

Edited by vegeta, 12 July 2011 - 11:28 PM.

[cashlern]

Yes, to ease your mind I would definitely make TAE with DEPC ddH20. And it never hurts to clean the plastic of everything i.e. comb, box, pipettes with RNAase zap. What I don't understand is why you heated your RNA samples at 60C. is that something you're supposed to do in the kit? I use trizol/acid phenol/chl/iosamyl to isolate my RNA, DNAse treat the RNA, and then do a second acid phenol/chl/iosamyl isolation.

[Trof]

Heating RNA in the presence of divalent cations can cause enzyme-independent degradation of the RNA.
We put formamide to the sample prior loading it to the agarose gel to denature RNA, instead of heating. We use the same tank and running buffer as for other aplications, never treat them special. Also I remember TAE gels heating themselves more than TBE gels we use, so maybe that could played a role?

On the other hand, just how bright was the smear in the first check? It could simply lost some fluorescence in time and become invisible if it was only a light smear.