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delaying RNA extraction; using buffer RLT to disrupt cells and place on ice?

RNA RLT PBMCs

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3 replies to this topic

#1 rumjon

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Posted 13 February 2013 - 04:28 AM

Dear all,

I am currently extracting RNA from circulating PBMCs.
My current experiment requires that I extract RNA at 10 minute intervals.
As it takes around 30 minutes for me to extract RNA (Qiagen RNeasy), it is clear that I really can't perform my experiment without some help.
I was wondering whether anyone had used buffer RLT to disrupt their cells, and then placed them on ice until a time when the rest of the RNA extraction protocol could be followed?
If not, then any other suggestions would be most welcome.

Many thanks in advance.

Edited by rumjon, 13 February 2013 - 07:44 AM.


#2 cellthetruth

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Posted 13 February 2013 - 06:32 AM

I suggest you perform TRIzol RNA extraction. Pellet the cells, add Trizol, homogenize, freeze in liquid Nitrogen. These steps should not take more than 10 min/sample. After you collected all your samples you can extract the RNA simultaneously.

#3 Tabaluga

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Posted 13 February 2013 - 07:20 AM

As far as I know it's possible to store the sample in RLT Buffer (after the lysis and homogenization) frozen at -80 C, although I think RNA recovery might be a bit lower than if you process them fresh (look it up ine the RNeasy manual, there is a paragraph there on freezing in the protocol). So would it be an option to store the samples till all of them are collected and then process them simultaneously ?
Or could you simply store the cell pellets ?

If in doubt, you can also ask the qiagen support what they think about putting the samples on ice...

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#4 rumjon

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Posted 13 February 2013 - 07:42 AM

Many thanks for your replies.





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