Ronit’s Notebook: RNA Extraction for C.Gigas Heat Stress Samples

I started working with the C. Gigas heat stress samples to extract RNA for gene expression analysis. Sam took care of 8 of the samples and I did the RNA extraction for the other 8 samples: D09, D10, D11, D12, T09, T10, T11, T12

The protocol I followed is as follows:

  1. 500 µL of RNAzol RT was added to a clean tube.
  2. Tissue samples were removed and a small section was cut out for RNA extraction.
  3. Tissue portions were placed in the tube and an additional 500 µL of RNAzol RT was added to bring the volume up to 1mL.
  4. The samples were vortexed vigorously for 10 seconds
  5. Samples were incubated at room temperature for 5 minutes.
  6. 400 µL of DEPC-water was added to the samples.
  7. Samples were centrifuged for 15 minutes at 12,000 g.
  8. 750  µL of the supernatant was transferred to a new, clean tube and an equal volume of isopropanol was added to the sample.
  9. The samples were vortexed vigorously for 10 seconds.
  10. Samples were incubated at room temperature for 5 minutes.
  11. Samples were centrifuged for 15 minutes at 12,000 g.
  12. The supernatant was discarded and 400 µL of 75% ethanol was added to the samples.
  13. Samples were centrifuged for 1.5 minutes at 4,000 g.
  14. Supernatant was discarded and an additional 400 µL of 75% ethanol was added to the samples.
  15. Samples were stored in -80 degree freezer.

Due to time constraints, we decided to finish up the extraction next week/quantify RNA using the Qubit and stored the RNA pellet suspended in ethanol in the -80 freezer.