RNA isolation problems - Have a look at my spectrum... (May/08/2006 )

Hi folks,

this really starts to annoy me: I isolate a lot of RNA from tissues using Trizol as a standard procedure, works perfectly, however today I tried the second time to isolate RNA from cultured fibroblasts (60.000 cells/tube). And the OD measurement looks really creepy. Some kind of double peak at 260 and 270nm. Its no protein contamination, as 280nm is not that high

Anybody ever seen something like that? What could be the reason? Any ideas?

Thanks a lot guys.....

And here is the spectrum (measured with Nanodrop in Tris 10mM pH7.5):

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hi
you definitely need to re-do steps from phenol/chlo and ethanol precipitate. Add eventually a carrier if quantity is sensitive, and let your sample air dry correctly. If you don't have enough time, heat it to 37° and no more for drying.

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Thanks.

I used sufficient amount of glycogen as carrier. All EtOH was removed before resuspension. Sure, I can repeat the precipitation, etc...

But does anybody know what the 270nm value is about, I have absolutely no idea. I never saw something like this before and I find nothing in the literature what this could be...

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I've found that on the net:

“Some reagents used in RNA purification protocols (TRIzol) can absorb at 230nm (also at about 270nm).
(source: www.bcm.edu/mcfweb/?PMID=3100)

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Yep, it's the organics, solvents and salts again. High 260/230 ratio = impure sample. Do what Fred said and re-purify

The "270 reading" you are talking about is the nucleic acids

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Thanks thats a good link... 270 nm reading as contamination from the Trizol reagent ist also interesting, I never heard about that.



I think high 260/230 ratio is good and low is contaminated...

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yes you're right. It's a little confusion.

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OK, I was (luckily) never forced to re-precipitate my RNA before. So my plan would be:

20 ul RNA in RNA secure (in 10mM Tris)
add 100 ul of H2O (RNAse free)
add 300 ul of isopropanol
incubate at -20°C for at least 20 min
spin
wash pellet 2x in 75% EtOH
resuspend RNA in RNA secure/Tris

measure again and hope for lower 230nm and 270nm...

Is that a good plan or should I change something?
I don't think that I have to repeat the whole Chlor/phenol extraction, as there is obviously no protein contamintation?

Ciao and thanks, Tobias

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hi
you have a 230 contamination. So it may comes with phenolic contamination.
So i would just add 280µl of RNAse free water,
add 300µl chloroform.
Shake vigorously for 30''
let sit 5' at RT.
Spin 12000g for 15' at 4°
Pipett the upper aqueous phase, add glycogen (1µl of a 100mg/ml) vortex gentle and briefly
add 300µl IprOH (to this point i would remind you that EtOH needs 2.5 x volumes, but the isopropanol needs only a 1:1 ratio)
Wash with 1ml EtOH 70%
let dry at 37° for 20-30'
resuspend, denature 10' at 56° and quantitate.
z

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Thanks for the quick reply :-) I will try your procedure (I thought somehow a simple precipitation would be sufficient to remove most of the phenol).

But I never denatured my RNA at 56°C before measurment. Is it really that beneficial? I have to do 10' at 60°C for the RNA secure activation, but always measured before this step. I could also easily measure after heating if this gives more consistent results...

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