RNA isolation - combination of Trizol & colum-based kit (Jun/28/2005 )
Is there any one using Trizol and column-based kit for your total RNA isolation? As far as I know, many people are using combination of Trizol method and column-based kit (such as QIAGEN RNeasy mini/midi kit) for their RNA isolation, specially when the isolated RNA is used for microarray.
And routine is that people iosolate total RNA using whole procedure of Trizol method, and then apply the isolate RNA to column in order to get cleaned RNA sample.
But I'm just wondering if extracted aqueous phase after first centrifugation (rather than dissolved RNA pellet obtained after final centrifugation) from Trizol method can be used as starting material for column-based kit (QIAGEN RNeasy) for total RNA isolation without processing further steps in Trizol method.
I don't know... never tried it myself. Try it and tell us what happens.
It is a really good idea and never thought of that myself... gotta stick that in the back of my brain for safekeeping.
I use trizol for RNA isolation, i take it you want to isolate using trizol then add chloroform to separate layers, then take the aqueous phase to a column, the next stages of trizol extraction simply include isopropanol ptt and a wash in 70% EtOH before resuspending pellet, is a kit worth the expense as most of the hard work has already been completed?
You have a point microman, but the difference would be that if you apply the aqueous phase to the column, you would avoid any DNA contamination and would not have to worry about losing the RNA pellet during the precipitation step.
i agree with microman. But i have a trouble. After the trizol extraction, usually it usually remains a few of phenol, doesn't it? and salts in the aequous phase can interfers with column fixation, can't it?
Honnestly i don't use columns because i'm not doing microarrays. but i'm questionning myslef.
Any comments are welcome.
Is DNA contamination an issue in an RNA isolation as you generally DNAse treat it - at least i always DNAse treat my RNA immediately, check integrity, aliqot and then freeze.
I really can't disagree with Fred or microman, but I still think it is a clever (if more expensive) idea.
Try it and see what happens, tell us.