RNA Extraction with Trizol plus QIAGEN RNeasy mini kit

April 20, 2011 10:45, Last update: May 09, 2011 15:05

For cultured cells (3 to 5.106 cells).

Detailed protocol

  1. Pre-warm PBS to 37 °C and keep Trizol at room temperature.
  2. Aspirate medium, rinse the cell GENTLY with 1 ml of PBS.
  3. Aspirate PBS, add 1 ml of Trizol
  4. Scrape cells and transfer to 2 ml Eppendorf. Phase lock Gel Heavy, 2.0mL
  5. Add 200 µl of chloroform to each sample and mix gently.
  6. Centrifuge at 14000 rpm for 15 min at 4 °C.
  7. Recover the supernatant into a new Eppendorf.
  8. Add 350 µl of ethanol 100% to each sample, mix.
  9. Transfer all the solution into the column (QIAGEN RNeasy mini Kit): 700 µl at a time.
  10. Centrifuge at 14000 rpm for 30 s at room temperature, discard the flow-through.
  11. Add 350 µl of RW1 Buffer
  12. Centrifuge at 14000 rpm for 30 s at room temperature, discard the flow-through.
  13. For each sample, mix 10 µl of DNase (RNase-free DNase Set-QIAGEN) with 70 µl RDD Buffer, add 80 µl of this mixture to each sample (add to the center of the column).
  14. Wait for 15 min at room temperature.
  15. Add 350 µl of RW1 Buffer to the column.
  16. Centrifuge at 14000 rpm for 30 s, discard the flow-through.
  17. Add 500 µl of RPE Buffer (RPE Buffer is supplied as a concentrate. Ensure that 220 mL ethanols are added to RPE Buffer before use)
  18. Centrifuge at 14000 rpm for 30 s, discard the flow-through.
  19. Add 500 µl of RPE Buffer
  20. Centrifuge at 14000 rpm for 30 s, change to Collection Tube (2mL, without caps)
  21. Centrifuge at 14000 rpm for 1 min, change to Collection Tube (1.5 mL, with caps)
  22. Add 12-37.5 µl of RNase-free water, centrifuge at 14000 rpm for 30 s (don't through away the filtrate).
  23. Add 12-37.5 µl of RNase-free water, centrifuge at 14000 rpm for 1 min.
  24. Keep the RNA in freezer at 20 °C.