Trizol followed by column purification - (May/26/2005 )
Trizol works well for total RNA isolation from tissue, followed by column purification. What kind of column purification should I use?
hi
standard RNA isolation columns are not suitable for small RNA isolation (siRNA for example). If you want to do that, centricon columns are ok to purifi special class of RNA.
standard RNA isolation columns are not suitable for small RNA isolation (siRNA for example). If you want to do that, centricon columns are ok to purifi special class of RNA.
Thanks!
but do i really need a column purification after Trizol treatment? Anyone who experiences this?
hi
i've never done column purification after trizol and my experiments are ok. Moreover, i think that colmns retains differntly short RNA than long RNA. Hence i rather not make a desequilibrium.
Of course we do need to purificate our total RNA! Since after the isolation genomic DNA will still in your isolate, it is very important to do a DNase treatment. Contamination of genomicDNA can be impact the true expression pattern in your sample.
I used to combine TRizol isolation with RNeasy column(Qiagen) along with AbsoluteRNA wash solution (invitrogen). Add -20C Eathnol to supernatant , then apply it to the RNeasy column. And go further according to the protocol.
A new product, TURBO DNase from Ambion, sounds more powerful, but I have not experience of this.
Good luck 
I just isolated total RNA using Trizol, the OD260/280=1.78. Then I used a traditional protocol to purify it, the OD260/280=2.16.
I have tried the RNeasy mini kit column to purify total RNA, however it does not work. If you would know the protocol, please contact with me.
I just isolated total RNA using Trizol, the OD260/280=1.78. Then I used a traditional protocol to purify it, the OD260/280=2.16.
I have tried the RNeasy mini kit column to purify total RNA, however it does not work. If you would know the protocol, please contact with me.
[/quote]
What do you mean 'it does not work'? In what kind of RNA isolate from you put on the column?
I know there is a maximum ug RNA you can put on the column. May be you can have a check.
Followed the protocol, I put 100ug RNA(OD260/280=1.78) on RNeasy mini column, however, I got back 10 ug RNA(OD260/280=1.85). maybe something was going wrong.If you have a protocol for it, please let me know. thank you!
quote=Ribosoul,May 31 2005, 02:07 PM]
I just isolated total RNA using Trizol, the OD260/280=1.78. Then I used a traditional protocol to purify it, the OD260/280=2.16.
I have tried the RNeasy mini kit column to purify total RNA, however it does not work. If you would know the protocol, please contact with me.
[/quote]
What do you mean 'it does not work'? In what kind of RNA isolate from you put on the column?
I know there is a maximum ug RNA you can put on the column. May be you can have a check.
[/quote]
[quote=likarlli,Jun 1 2005, 05:41 AM]
Followed the protocol, I put 100ug RNA(OD260/280=1.78) on RNeasy mini column, however, I got back 10 ug RNA(OD260/280=1.85). maybe something was going wrong.If you have a protocol for it, please let me know. thank you!
I also use RNeasy mini column for purification, and the offer is ~20% of my RNA. I think there is something going wrong as you loos so much of your RNA.
Here is the protocol, it must be supplied along with the kit.
http://www1.qiagen.com/literature/handbook...NY_062001WW.pdf
read:RNeasy Mini Protocol for RNA Cleanup on page.79
don't forget to add β-Mercaptoethanol (β-ME) must be added to Buffer RLT before use.
The same to Buffer RPE with ethanol (96–100%). And you have to realize that if your kits has already been used for a period, the ethanol may evaporate, that mean you may change a new kit.
Hope this help 
PS. May I know how is the RNA perserved, in what solution and under which temperature?These all many also be a reason.
total RNA is fine(store at minus 80 degree, in RNase free water), I have checked by running gel. Maybe the problem is that I forget to add β-Mercaptoethanol (β-ME) to RLT buffer. Thank you very much!
[quote=Ribosoul,Jun 2 2005, 12:56 PM]
[quote=likarlli,Jun 1 2005, 05:41 AM]
Followed the protocol, I put 100ug RNA(OD260/280=1.78) on RNeasy mini column, however, I got back 10 ug RNA(OD260/280=1.85). maybe something was going wrong.If you have a protocol for it, please let me know. thank you!
I also use RNeasy mini column for purification, and the offer is ~20% of my RNA. I think there is something going wrong as you loos so much of your RNA.
Here is the protocol, it must be supplied along with the kit.
http://www1.qiagen.com/literature/handbook...NY_062001WW.pdf
read:RNeasy Mini Protocol for RNA Cleanup on page.79
don't forget to add β-Mercaptoethanol (β-ME) must be added to Buffer RLT before use.
The same to Buffer RPE with ethanol (96–100%). And you have to realize that if your kits has already been used for a period, the ethanol may evaporate, that mean you may change a new kit.
Hope this help
PS. May I know how is the RNA perserved, in what solution and under which temperature?These all many also be a reason.
[/quote]