RNA pellets seem too big: normal or something else in there? - (Feb/28/2006 )
I have been extracting RNA using the TRIzol method from primary human hepatocytes, and my pellets are very large and white, not clear and gel-like. When I measure A260, I get yields as high as 200 µg from 1/2 of a 60-mm dish. 260/280 is usually around 1.7 to 2.1. I don't have any experience other than using these primary cell lines, but I heard recently that yields that large are not "normal". In addition, I have been using Qiagen columns to purify the RNA after extraction, and I get NO RNA following column clean-up. I have been working with Qiagen tech support for weeks and they are starting to think that my A260 readings are not from RNA; however, Bioanalyzer results indicate that decent quality RNA is present. But now I wonder if I am clogging the column with whatever else is in that huge pellet?? Does anyone know what else might be in there, or is it possible it really is all RNA??
(By the way, I'm working on my supervisor to let me change purification methods, but as we've already started a contract project with this method, we have to see it through for now.)
Any advice, suggestions, or comments are welcome. Thanks a lot!
It doesn't sound as though your quantity of RNA is that way off. I routinely get about 30 µg from 3 wells of a 12-well plate. This can shoot up to 100 µg per 3 wells (i.e. about 400 µg per plate) when the cells are induced.
As for the Qiagen problem. I know someone else whp has had this problem. Not too sure what is going on there. Ask Qiagen to send you new columns and buffers from a separate batch.
Oh, and to get nice gel-like pellets you have to wash them a load of times in 70% EtOH. Sometimes 4-5 times until the whiteness starts disappearing.
Thanks for your reply. Interesting that other people have had this problem with the Qiagen columns because I honestly can't figure out what I'm doing wrong with them, if anything.
Also, if several washes are necessary to remove the white from the RNA pellets, does that mean that the "white stuff" is not RNA and is therefore a contaminant? Or do multiple washes simply reduce the RNA concentration so that the pellet no longer appears white?
doesn't it relate to the solvent used to precipitate, like with DNA? I know that EtOH/oAc ppt's of DNA are usually white and sort of fluffy; ISOH ppt's are usually translucent...I would guess it depends on what is used to wash the pellet...but perhaps it doesn't work like DNA?
As far as large yield being normal or abnormal...it's always different. depends on the cells, and on the treatment you give them....when I stimulate my little keratinocytes with some treatments vs others, I see changes in the overall yield. I make a note and see if it happens when I'm repeating the experiment (generally it does), but I don't think it's something to worry about.
it's all so damn subjective, that just makes it harder...we all do things a little differently, and that affects things like yield
high amount of salts results in white pellet. Several washes with etOH 70 drive elimination of them...
For add : this is particulary the case if you add glycogen to precipitate. But here again several washes are the solution to eliminate most of the glycogen. The remaining glycogen doesn't interfers with downstream exp.
Hi everyone, thanks for your replies!
Aimikins, it is good to know that there is no "normal" or "abnormal" yield of RNA. That does make sense, but since I have only worked with this system I wanted to make sure.
Fred, you have helped me with this problem both in this thread and in the one where you posted the TRIzol manual. A little while ago, I was discussing whether it is ok to store RNA overnight at -20C to precipitate after the isopropanol step. That manual says that I should NOT ppt the RNA at -20C because it also causes salts to ppt! That could explain my white pellet - although that manual also says that RNA pellets can be white.
I've been using TRIzol for a while, but I thought the only literature available was the product insert. Thanks for drawing my attention to the detailed manual.
hem... you're welcome
Purifying RNA's wihtout the success i hoped (siRNA depserately went out) i went trhough detailed Phenol chlo and trizol protocols to see what can help. You can phone invitrogen if you are puzzledafter searches...