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

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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

-gogreen-

So we do the RNase treatment using the Qiagen RNeasy DNase ...digesting DNA at 25C 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

-pDNA-

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.ac.uk/~toxo/Protocols/Protocols.html

-sanjiun-

@ 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!!!

-gogreen-

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.

-sanjiun-

@ sanjiun,

If the number of samples are less, you can go high on the extraction cost..but when you have too many samples, say a hundred or more, the savings from this can account to the cost of a couple of arrays itself !! :lol:

-gogreen-

yes, but, how much less does a failed array cost?

it is worth some extra expense to ensure the results of a microarray rather than the expense to repeat it.

and, what about sample availability? some samples are very precious. they may not be available to repeat the procedure.

there can be many reasons given to save money but if you can afford to perform an expensive test then you can afford a little bit more to ensure the quality of the sample being tested.

-mdfenko-

Trizol actually works for every kind of tissue and cells and in my hands I've done well over 500 arrays with RNA from bacteria and yeast to plants and clinical samples without a single failure if the purity and the integrity factors were good enough to proceed to labeling and hybs..At the scale at which I was working, trizol was working out incredibly cheaper compared to the Qiagen/Ambion kits !!

-gogreen-

gogreen on Nov 17 2009, 07:11 AM said:

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


i meant DNase treatment of RNA

-susanna-

Do you guys know this paper published in 2009 titled as 'A simple and loss-free method to remove TRIzol contaminations from minute RNA samples'?
At least I observed that the RNA purity increased a lot after purification following this simple and cheap protocol.

Link is below;
http://www.sciencedirect.com/science?_ob=A...5f0ccbcb7e882e4

-huni-
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