isolation of RNA from samples stored in RNA later (ambion) - can we use Qiagen total RNA isolation kits (Feb/28/2006 )
Hi
I have used RNA later to store some of the cells that I needed to extract RNA from
at -80C
I would like to know if anyone has had success isolating RNA from cells stored in RNA later and how did they get back their cells from RNA later before proceeding with the Qiagen kit
thanks
-Watson-
There is a protocol in the RNAlater manual for re-pelleting your cells. Something like adding an equal volume of PBS then centrifuging briefly
When using RNAlater to store cells I have always experienced some loss in yield, but the integrity is always fine
-John Buckels-
QUOTE (John Buckels @ Mar 1 2006, 04:37 AM)
There is a protocol in the RNAlater manual for re-pelleting your cells. Something like adding an equal volume of PBS then centrifuging briefly
When using RNAlater to store cells I have always experienced some loss in yield, but the integrity is always fine
When using RNAlater to store cells I have always experienced some loss in yield, but the integrity is always fine
thanks a lot
just one more clarification
once I add the PBS and then spin the cells briefly.. I will get a pellet ...then I can resuspend this pellet in the lysis buffer from qiagen RNeasy mini kit...is tha what you had meant
thanks a lot..again
-Watson-
Yes, that is correct
manual here
QUOTE
Cells
There are two options for isolating RNA from cells stored in RNAlater: The preferred method is to remove RNA later from the cells prior to extraction. Alternatively, cells in RNA later can be used directly for RNA extraction. Because of the greater volume that the cells are in, this method generally requires additional lysis solution.
• Removal of RNAlater prior to extraction
Because of the density of RNAlater, greater cetrifugal forces are required to pellet cells from RNAlater than normal media. Generally, cells become much less fragile when stored in RNAlater and can be centrifuged at high speed without lysis. Most cell types can be centrifuged at 5000xg without damage to the cells. Since different cell types vary in their ability to withstand centrifugal forces, we recommend testing the centrifugal speed with an expendable sample. Alternatively, dilute the RNAlater by adding an equal volume of ice cold PBS (or other buffered
solution) immediately before centrifugation to reduce the density of the solution, then centrifuge at normal speeds.
• RNA extraction from cells in RNAlater
One-step phenol-based disruption/extraction solutions (e.g., RNAWIZ ™ or TRI Reagent®) can be used to purify RNA from cells suspensions in RNAlater. This can be done by adding ten volumes of the one-step solution to the cell mixture, and proceeding normally. When Ambion’s RNAWIZ is used in this way, it may be necessary to dilute the aqueous phase before the RNA precipitation step, see below for more information.
There are two options for isolating RNA from cells stored in RNAlater: The preferred method is to remove RNA later from the cells prior to extraction. Alternatively, cells in RNA later can be used directly for RNA extraction. Because of the greater volume that the cells are in, this method generally requires additional lysis solution.
• Removal of RNAlater prior to extraction
Because of the density of RNAlater, greater cetrifugal forces are required to pellet cells from RNAlater than normal media. Generally, cells become much less fragile when stored in RNAlater and can be centrifuged at high speed without lysis. Most cell types can be centrifuged at 5000xg without damage to the cells. Since different cell types vary in their ability to withstand centrifugal forces, we recommend testing the centrifugal speed with an expendable sample. Alternatively, dilute the RNAlater by adding an equal volume of ice cold PBS (or other buffered
solution) immediately before centrifugation to reduce the density of the solution, then centrifuge at normal speeds.
• RNA extraction from cells in RNAlater
One-step phenol-based disruption/extraction solutions (e.g., RNAWIZ ™ or TRI Reagent®) can be used to purify RNA from cells suspensions in RNAlater. This can be done by adding ten volumes of the one-step solution to the cell mixture, and proceeding normally. When Ambion’s RNAWIZ is used in this way, it may be necessary to dilute the aqueous phase before the RNA precipitation step, see below for more information.
-John Buckels-