Ronit’s Notebook: RNA Extraction for Remaining C. Gigas Dessication + Elevated Temperature Samples

I extracted RNA for 16 samples (D05, D06, D07, D08, D15, D16, D17, D18, T05, T06, T07, T08, T15, T16, T17, T18). RNA pellets were stored in the -80 freezer following isopropanol precipitation. The protocol I used is described below:

  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. Note: one of the RNA pellets (D17) looked black. Not sure what could have caused this or if this is a sign of contamination, but I’ll proceed with the RNA extraction for D17 and see what the Qubit results show for that sample.