delaying RNA extraction; using buffer RLT to disrupt cells and place on ice? - (Feb/13/2013 )
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.
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.
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...
Many thanks for your replies.