RNA Gel Problems - RNA not showing up as bands in gel (Oct/20/2005 )

I'm reading up on ways to optomize my RNA gels.

Invitrogen recommends pre-running the gel before loading RNA samples. Why? Does this actually help?

Some protocols say it is optional to add EtBr to the loading buffer. Others recommend adding it to the electrophoresis buffer. I have added it to the gel only, shouldn't this be enough? I'm just looking to check the integrity of my RNA, not to extract it afterward.

I always added EtBr to both the RNA loading buffer and the gel, I think this improves the sensitivity. The main concern is that EtBr will change the migration pattern of the bands, possibly lead to spreading, but for assessment of quality there is no reason not to add it... are you running formaldehyde gels?

I am currently doing formaldehyde gels, though after I get my RNA working, I would be content to use native agarose gels just to check my bands and make sure my RNA extractions were ok.

I've done numerous gels on numerous samples of extracted human RNA from cell culture, using Trizol and RNAeasy. The spec reads RNA.

My gels, on the otherhand, don't agree. This past gel I ran- I got NOTHING at all. I added EtBr into the denaturing loading dye. Maybe I didn't use enough RNA? The manufacturer's protocols said 3 ug = 1 uL for me.... (and matching part loading dye, = 1uL, total 2 uL.)

But before this, all I was getting was a faint smear down the entire lane. Looked like all the RNA had degraded.

And once- i actually saw a band... though it was very faint. And.... uh... only one band. there should have been 2.

Hi,

I do not prepare formaldehyde gel for checking the RNA integrity. I run usual DNA agarose gel. I normally prepare %1 agarose gel with TAE buffer (this should be autoclaved) then add EtBr.Then I prepare my sample like this:

for example: 5 ul RNA+ 1 ul 6x DNA loading dye+ 6 ul formamide

then I load it on gel and run at 70 V for 30 min.

I attach one of my RNA gel photos

Hi,

I do not prepare formaldehyde gel for checking the RNA integrity. I run usual DNA agarose gel. I normally prepare %1 agarose gel with TAE buffer (this should be autoclaved) then add EtBr.Then I prepare my sample like this:

for example: 5 ul RNA+ 1 ul 6x DNA loading dye+ 6 ul formamide

then I load it on gel and run at 70 V for 30 min.

I attach one of my RNA gel photos

The DNA loadying dye won't degrade the RNA?

I did not see any degradation on the gel:)as far as I know RNA loading buffers contain almost the same reagents with DNA loading dyes but additionally there is only formamide in it for denaturation of RNA. Look at this page it can give you an idea about RNA loading buffers :

http://www.fermentas.com/catalog/electroph.../rnaladders.htm

See you...