RNA samples degraded in gel... where is the source of contamination? - (Jan/24/2008 )

I had sucrose gradient fractioned.
The sucrose buffer contains...
Heparin, RNaseOut, cyclohexamide.

The sample was not denatured, so there must be RNase in the sample???
(even though most of proteins are in the top fraction which was not tested this time)

25ul of samples were mixed with 5ul of Formaldehyde (no formamide),
(and 1ul of 1.67mg/ml Ethidium Bromide),
heated 70C for 15 minutes. 6ul of loading buffer was added.
Then they were run in 1.2% gel with Formaldehyde .
After the formaldehyde and heating, RNase in the sample is denatured and deactivated, right?

Gel box was soaked with 10mM NaOH + 0.1% SDS for a week.
Lid for the box was cleaned with RNaseOff.
Flask used with aluminum foil was also treated with NaOH/SDS.
Comb was cleaned with RNaseOff.
All the water used was DEPC treated.

I attached the gel I got.
Last two lanes were supposedly intact RNA.
They were completely degraded.
Other's are samples from sucrose gradient fractions which still have Heparin and RNaseOut...
I guess that is why I can still see clouds of two subunit??? is it?

Do you have any suggestion for improvement?

to me it occurs like there is a problem with the gel itself rather than the RNA... next time you could load a ladder to check if the gel has run correctly.

What I would do is to run a regular agarose gel, use 72µl formamide+36µl glycerol+1µlBromophenol Blue as loading buffer. You should use 10µl of loading buffer per 1µl of sample you'd like to use. just load it without heating. I run at 50V for 1 h and 75V for 15 minutes but basically you run until it has run enough. This should be adequate to check if your RNA is intact or not.

Thank you very much!!!! I will try that and report what happens.

No worries, I am also curious now:))

Oh, 1 hour and 15 minutes! That is great. I can go back home early
Yes, I just need to separate those two subunits.
So the addition of formamide keeps the RNA from degrading by denaturing possible contaminating RNase?
I am going to pour gel tomorrow morning and then run the gel with only supposedly intact RNA samples.
I also got relatively newer gel box from our neighbor, so I will use that, too. I hope it goes well.

"So the addition of formamide keeps the RNA from degrading by denaturing possible contaminating RNase?"

nope, it stabilizes the RNA in the gel by deionizing it.

Good luck!

Finally!!! I got intact bands with formamide. Sample without formamide was degraded even though it was run on the same gel. I also ran samples with GelRed since the Ethidium Bromide stock was more than a decade old. I thought it could be the source of contamination. Curiously the EB sample was ok but GelRed did not have anything on the gel... at least not detected. I thought it was EB alternative and I can just use the UV to excite and EB filter to detect. Anyway...

Thanks a lot for your suggestion. You saved a lot of my time.

Hey! I am glad that it worked:)) The thing with GelRed can be that the exposure time is way longer than EtBr, so next time you don't see any band just expose longer. It should be there. I use SYBR Green II. very sensitive. it's just a pain to change the transilluminator:(

You did that, right? I mean you changed the transilluminator before looking at the gel with GelRed??

And don't be so paranoid with RNAase contamination, if you extracted your RNA clean, it should not degrade so fast in the gel even if there's contamination.

Cheers!!

Oh... I needed longer exposure. I see.
I used UV transilluminator with EB filter. So that should be ok, right?
The protocol says

"View the stained gel using a standard transilluminator (312 nm)
and photograph the gel using Polaroid 667 films and an ethidium bromide filter."

They should say in the protocol "You need @!#$% long exposure time."

Yes, I think RNase contamination is driving me crazy. So I am relieved by what you said.

Thanks for your help, emiliania.

All right, my mistake. they do use the same transilluminator. Geez i have no idea, I had some problems visualizing my gels, too:(