Roche MagNA Pure 96 - Setting up RNA Purification

Main Applications
Setting up a Run for Purification of Cellular RNA from Cultured Cells
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Preparing the Samples
2 Setting up a Run for Purification of Cellular RNA
from Cultured Cells
This procedure describes the purification of cellular RNA from1x10
6
cultured cells resuspended in
200 µl PBS using the MagNA Pure 96 Cellular RNA Large Volume Kit.
Do not use more than1x10
6
cells. If necessary, count the cells prior to the purification run. If the cell
number should be too high, dilute the cells using PBS.
Setting up a run for purification of cellular RNA comprises the following steps:
1. Preparing the samples and the DNase solution, see next section.
2. Defining the parameters in the software, see section Defining the Parameters in the Software.
3. Loading the instrument, see section Loading the Stage.
2.1 Preparing the Samples
The following procedures describe the treatment of standard cell lines in a 75 cm
2
cell culture flask. De-
pending on the characteristics of the specific cell line, specific parameters of the procedures might be dif-
ferent, e.g., centrifugation force, volume/concentration of trypsin.
Always use sterile, nuclease-free pipettes.
To prepare adherent cells
Visually check the source plate to ensure that the samples are pipetted into each individual well of the
source plate.
Take the cell culture flask or plate out of the cell culture incubator.
Remove the cell culture medium from the cell culture flask or plate.
Add 8 ml PBS to wash the cells.
Remove PBS from the cell layer.
Optionally repeat steps 3 and 4 to wash the cell layer twice.
Add trypsin and incubate for 2 minutes in the cell culture incubator.
To trypsinize a cell layer of a 75 cm
2
plate, use 2 ml trypsin solution (conc. 0.05%).
Check under the microscope if the cells are detached. If cells are not detached, continue incubation in
the cell culture incubator and check every minute whether they are detached.
Add 8 ml PBS to the cells resuspended in trypsin and shake the cell culture flask or plate slightly.
Transfer the cell suspension into a suitable centrifugation tube.
Centrifuge the cells for 10 minutes at 300 x g.
Discard the supernatant and resuspend the cell pellet in 200 µl PBS.
Pipette the 200 µl cell suspension into the individual wells of a processing cartridge. This cartridge is
then defined as the source plate.
Pipette the samples into the bottom of the wells using aerosol-preventive tips.
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