RNA extraction from cells cultured on ACCs

We have tried to perform RNA extraction from MSC growing and differentiating cells using several protocols. The protocol described here is the adaptation from the manufacturer’s instructions with which we were able to successfully extract RNA from MSC cells growing and differentiating on a carbon cloth matrix.

Commercial kit used: Purelink RNA Mini Kit (Life Technologies, 12183018A). Manufacturer’s full protocol is available on their webpage.

Lysis and Homogenization of cells on ACCs

  1. Remove the growth medium from the wells.
  2. Add 700 µl of Lysis Buffer with 2-mercaptoethanol freshly added, vortex.
  3. Transfer the lysate to a 1.5 mL RNase–free tube and pass 5–10 times through the pipette tip.

Binding, Washing, and Elution

  1. Add one volume of RNAse free 70% ethanol
  2. Vortex to mix thoroughly and to disperse any visible precipitate that may form after adding ethanol.
  3. Transfer up to 700 µL of the sample (including any remaining precipitate) to the Spin Cartridge (with the Collection Tube).
  4. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through, and reinsert the Spin Cartridge into the same Collection Tube.
  5. Repeat Steps 3–4 until the entire sample is processed.
  6. Add 700 µL Wash Buffer I to the Spin Cartridge. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through and the Collection Tube. Place the Spin Cartridge into a new Collection Tube.
  7. Add 500 μL Wash Buffer II with ethanol to the Spin Cartridge.
  8. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through and reinsert the Spin Cartridge into the same Collection Tube.
  9. Repeat Steps 7–8 once.
  10. Centrifuge the Spin Cartridge at 12,000 × g for 1-2 minutes to dry the membrane with attached the RNA. Discard the Collection Tube and insert the Spin Cartridge into a Recovery Tube.
  11. Add 30 μL–3 × 100 μL RNase–Free Water to the center of the Spin Cartridge.
  12. Incubate at room temperature for 1 minute.
  13. Centrifuge the Spin Cartridge for 2 minutes at ≥12,000 × g at room temperature to elute the RNA from the membrane into the Recovery tube.
  14. Proceed to Analyzing RNA Yield and Quality on nanodrop and on a non-denaturing gel.
  15. Store at -80ºC.

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