RNA extraction for microarray - (May/13/2009 )

Hey everyone,
I am wondering if RNA extraction by trizol is compatible with the microarray analysis (trizol extraction method with no further purification)?
Thanks in advance
have a nice day
sam

If I remember correctly, affymetrix recommends the purification of RNA via columns after the use of trizol for rna isolation.
column isolation of rna via columns (RNeasy or something similar) usually gets rid of DNA contaminations as well to a very high extend if you do not overload columns.
However, I know from colleagues who just did a trizol extraction without further purification afterwards.

thanks for the reply..
ya actuelly I am wondering if I can do just like these people as I don't have much of RNA and using the kit I will lose a part of RNA
thanks again
Regards

Well, I can only tell you from my colleqgues that their arrays worked, but that might also strongly depend on the platform you take - we are mainly using affymetrix here which is probably very robust.
How much RNA do you have?

Hi

using only Trizol extraction means there are still phenol / salts present in your sample.
This means that or the labeling of your RNA will not be optimal OR/AND the stringency of your hybridization conditions will drop because the phenol is saturated with salt.

So in my opinon you should perform a precipitation but better would be a silica column purification after the phenol/chloroform extraction.

phillip

Hi, i also have same doubt here. I was isolating total RNA from whole blood using tri-reagent method. In fact, i only can get 0.226ug/ul of concentration from 250ul starting material of whole blood. Since i need at least 10 ug total rna to start my microarray, should i pool down my rna from aliquots and re-precipitate? or is it safer i perform spin column method first? All i need to do is to make sure i can get enough yield and pure RNA for a successful microarray.

Really need your advise. Thanks.

@wntiong

what type of microarray do you use?

We do first Trizol extraction, than get rid of the DNA by DNase I digestion followed by Phenol:Chloroform:Isoamylalcohol extraction ...than the DNA should be pure enough to use it for genertion of cDNA by RT. This works great for bacterial RNA because we have loads of them and don't have to worry that we lose some amount of RNA during the two extraction steps.

One thing you should think about when using columns for purification ...you might lose all transcripts that are smaller than for example 200 bp, because they won't bound to the matrix of the column ...so this information will be lost! Therefore we prefere using classical methods.

Hope this will help you somehow!

Regards,
pDNA

in my opinion,you should purify RNA and treat it with DNAse before a microarray experiment....if you have less quantity of RNA,you can always amplify it.there are enough kits available which will allow you to amplify rna and then use it for labeling (Nugen is a company offering one).you can even do a microarray with ng quantities of rna. but if you dont purify it, then there might be a problem labeling it.

pDNA on Nov 8 2009, 04:38 AM said: