25 replies to this topic

[samsaf]

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

[Bomber]

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.

[samsaf]

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

[Bomber]

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?

[ph1ll1ps]

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

[wntiong]

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.

[Calypso]

@wntiong

what type of microarray do you use?

[pDNA]

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

[Helios]

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.

[susanna]

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

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

And is trizol extraction also good when you will do the DNA-ase treatment of DNA, using filter units, like millipore? and for the application of qPCR?

with kind regards,

[gogreen]

Susanna, U asked about giving DNase treatment to DNA!!!

I suppose it was DNase treatment for RNA..but I didnt get which method of DNase treatment u were refering to here! You can do a trizol extraction, then do a DNAse digestion on column or use the Ambion Turbo DNase to avoid a column cleanup step where you lose the small RNA fraction

[pDNA]

So we do the RNase treatment using the Qiagen RNeasy DNase ...digesting DNA at 25°C for 10 mins. But in general the TRIZOL extraction does not interfere with any method as far as i know! The Ambion page has good technical references on the work with RNA if you are interested!

Regards,
              p

[sanjiun]

Something to share:
I found this a very good/ clean protocol. I used to use this to extract human cell line RNA for microarray.

http://www.path.cam..../Protocols.html

[gogreen]

@ sanjiun - The protocol uses Trizol and Qiagen columns for the extraction!! If you belong to a lab which is not "quite well funded", this might be expensive for a large number of samples.

I  had been using normal Trizol extracted RNA without a DNase cleanup for microarrays for a long time without any problem. I would choose Trizol over a column because its quite inexpensive compared to column, quite robust and has a very vast range of starting amounts of cells/tissue which can be used (100s to >10^7 per ml of Trizol) and the best part, it works for all the kinds of stuff, be it cell lines, human tissues, plants or yeast!!!

Edited by gogreen, 18 November 2009 - 07:09 AM.

[sanjiun]

gogreen, on Nov 18 2009, 11:05 PM, said:

@ sanjiun - The protocol uses Trizol and Qiagen columns for the extraction!! If you belong to a lab which is not "quite well funded", this might be expensive for a large number of samples.

True.
I use that cause my samples are not too many.
Since microarray itself is already an expensive experiment. LOL
So my supervisor wanna make sure everything worked well and get good RNA for microarray.