Guidelines for Purification of RNA from Cultured Mammalian Cells
- RNA can be purified from as few as 100 cells up to as many as 1x107 cells in a standard prep.
- Proteinase K digestion is not required for processing cultured mammalian cells.
- Monarch DNA/RNA Protection Reagent is not required for this protocol. Cells are lysed in the RNA Lysis Buffer.
- For cells stabilized in DNA/RNA Protection Reagent, allow samples to equilibrate to room temperature prior to the addition of an equal volume of RNA Lysis Buffer.
- If working with frozen cell pellets, thaw briefly (30-40 seconds at room temperature) prior to resuspension in RNA Lysis Buffer.
- RNA Lysis Buffer volume recommendations are for pelleted cells, however, cells in suspension can be directly processed by adding 4 volumes of RNA Lysis Buffer.
- After lysis, do not place samples on ice. Keep samples at room temperature to prevent precipitation of detergent in the lysis buffer. All subsequent steps should be performed at room temperature.
- Adherent cells can be lysed directly in the wells of a multi-well plate.
For immediate processing:
A. Remove the media from the well and rinse with PBS.
B. Add Monarch RNA Lysis Buffer (NEB #T2012) diretly to the well according to the table below:
Multi-well plate size Volume of Monarch RNA Lysis Buffer 6 well > 600 μl 12 well 300 - 600 μl 24 well 300 μl 48 well 300 μl
A. Remove the media from the wells and rinse with PBS.
B. Add 1X Monarch DNA/RNA Protection Reagent (NEB #T2011) (be sure to dilute the 2X concentrate) directly to the well according to the table below:
Multi-well plate size Volume of 1X Monarch DNA/RNA Protection Reagent 6 well >600 μl 12 well 300 - 600 μl 24 well 300 μl 48 well 300 μl
D. When ready to process, add an equal volume of Monarch RNA Lysis Buffer (NEB #T2012) and proceed to Step 1 of Part 2: RNA Binding and Elution.