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Milky solution after adding 100% ethanol - RNA extraction (Mar/08/2007 )

Hey guys,
I am having degradation issues with RNA. During RNA isolation, after adding chloroform and spinning, collecting the aqueous layer, when i add absolute ethanol, the solution is turning in to a milky cloudy solution. What could be the reason. I use trizol. I usually add excess of ethanol so that all the tubes have equal amounts of solution and i can centrifuge all of them at a time (yeah i am lazy). In doing so, tubes having low quantity of aqueous solution might get excessive volumes of ethanol. Is it the cause? Please do reply.

Thanks in advance.

-polsum-

QUOTE (polsum @ Mar 8 2007, 12:44 PM)
Hey guys,
I am having degradation issues with RNA. During RNA isolation, after adding chloroform and spinning, collecting the aqueous layer, when i add absolute ethanol, the solution is turning in to a milky cloudy solution. What could be the reason. I use trizol. I usually add excess of ethanol so that all the tubes have equal amounts of solution and i can centrifuge all of them at a time (yeah i am lazy). In doing so, tubes having low quantity of aqueous solution might get excessive volumes of ethanol. Is it the cause? Please do reply.

Thanks in advance.


You must have large amount of proteoglycan or polysaccharide in the tissue you are working with, like liver and kidney. The milky solution you got is mainly caused by precipitation of proteoglycan or polysaccharide.

The solutions to your problem are:

1). Use more trizol when you work with those tissues. Usually you add 1ml trizol per 100mg tissue, right? Try 2ml trizol per 100mg tissue instead.

2). Or you can precipitate your RNA by adding 0.25ml of (0.8M sodium citrate and 1.2M NaCl) per 1ml of starting trizol, then 0.25ml of isopropanol. It is writen in the manual of the Trizol reagent.

-MappleBrook-

Thanks mappleBrook,

I actually added more trizol this time and extracting rna. Let us see how the quality turns out.

-polsum-

Ok,

The RNA quality seems to be Ok. Atleast i dont see any degradation of rna. But because i used excess trizol, 260:230 ratio was very low. So i added chloroform and collected supernatant and added 100% ethanol. This time the solution is much more milky and i saw a big white precipitate immediately forming after i added ethanol. I extract rna from mouse lung tissue. The lungs had inflammation. Could anyone tell me why i still see the milky solution and instantaneous precipitation?

thanks in advance.

-polsum-

milky solution is seen probably because you have great amount of RNA.
How is your conc after your first prep?

also for getting higher Ratio 260:230, wash other time with 70%ethanol.

-fred_33-

QUOTE (fred_33 @ Mar 15 2007, 10:52 AM)
milky solution is seen probably because you have great amount of RNA.
How is your conc after your first prep?

also for getting higher Ratio 260:230, wash other time with 70%ethanol.


I think you are right Fred. My samples have huge amounts of RNA. The concentration was well over than 1mg per ul.
To lower 260:230 ratio, i reprecipitated with 90% ethanol. The ratio improved, but there was a substantial loss of RNA.
Also, as i am using excess trizol, i see precipitated salts mixed with the rna pellet. I had hard time dissolving the pellet in rna. I literaally had to break the salt with pipettte tip. I could hear the crackling sound. Those salts actually deterred a proper electropherogram. I am planning to remove those salts with dialysis. I have no clue how its going to work. Why does the solution turn milky if there is excess of rna? Thanks for the reply.

-polsum-

hi
i would pellet the salted RNA using butanol. This is the more hydrophobic alcool routinely used. Then after this pelleting, you can do several washes of RNA in 70% ethanol.
If you have time, do an incubation in 2h, and pellet.

It's assumed that cracking the pellet is not necessary a critical step in desalting. Resuspending the pellet (and you can use a tip to help it) should be enough to dessalt.
Do several steps of wash should be good.
Also i don't have much clues, but i think that your ratio decreases in part due to loss of salts (but the question is : does salts absorb at 260 ?)

Finally, the milky solution comes from the fact you have plenty of RNA. As soon as you add ethanol, a part of the RNA start to form aggregates. These are visible and transform a clear solution in a milky one smile.gif
Also 1mg/┬Ál seems far too much to me.
How did you measure it (quantity, dilution fator, OD values...) ?

-fred_33-