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Mytilus Live Tissue Tri-Reagent RNA Extraction

Extracting RNA from Live Mytilus edulis tissue using a Tri-Reagent/Direct-zol RNA Isolation Protocol written by Johanna Harvey

Zymo Research Direct-zol RNA Miniprep Plus Kit

Prep

Doing 4 samples as a test with Amy Zyck

MOST STEPS DONE IN HOOD and wear lab coat

  1. Aliquoted 4mL of Tri-Reagent (stored in 4 degree fridge) into a 5mL tubeand kept in and ice bucket
  2. Aliquoted 400µl of 1-Bromo-3-Chloropropane (stored in the acid cabinet) into a 1.5mL tube and kept in and ice bucket
  3. Cleaned RNA bench-top and a new piece of foil with 10% bleach, DI H20, 70% EtOH, then RNaseZap. Did this between each mussel
  4. Put 4 mussels in a holding tank
  5. Clean RNA bench forceps and scalpel with DI water, 70% EtoH, RNaseZap, then Nuclease free H20 for each mussel
  6. Used fresh scalpel blade and cut open mussel through their hinge and around the slit between the two shells until I could easily pull it apart
  7. A few millimeter section of tissue was taken from the mantle and lightly chopped with the scalpel blade before being placed in a new 1.5mL tube
  8. 1mL of Tri-Reagent was immediately added in the hood to each sample
  9. Beads were cleaned with RNaseZap then isopropanol, then put one each in the sample tubes
  10. Tissue was homogenized by taping the 1.5mL tubes closed and securing them on the plate-vortexer shaking at top speed until all tissue was determined homogenized visually. Shaking times needed varied per sample, but all samples were vortexed for the same amount of time
  11. All liquid was aspirated into a new 1.5mL tube
  12. Waste tubes and tips were disposed of in the bag in the hood for hazardous waste
  13. 100µl of cold 1-Bromo-3-Chloropropane was added to each sample tube and tubes were inverted repeatedly for at least 20 seconds. The liquid should look like pepto-bismol
  14. Incubated tubes at room temp in the hood for 15 minutes
  15. At this time turn on the centrifuge, set it for 2 degrees C and let it spin to get cooling down
  16. Centrifugation separates out the aqueous layer (with RNA) and the organic layer. Before centrifugation some separation of layers was already present
  17. Centrifuged all samples at top speed for 30 minutes at 2 degrees C
  18. Very carefully take the sample tubes out of the centrifuge as to not disturb the layers, and take back to the hood
  19. With a p200 pipette, remove ~500µl of the clear supernatant and put it in a new 1.5mL tube
  20. Tubes containing the organic phase were stored in the -80 for potential DNA extraction
  21. Added equal volume (~500µl) of 100% EtOH to the new sample tubes and inverted to mix
  22. Transferred 500µl of the liquid into the spin columns from the Direct-zol kit
  23. Centrifuged at 16,000 rcf for 30 seconds at room temp
  24. Transferred column to new collection tubes, added the other 500µl of sample, and repeated the spin after each spin samples were moved to new collection tubes
  25. Added 400µl of RNA Wash Buffer and centrifuged for 16,000 rcf for 30 seconds
  26. Calculated volumes for DNase I treatment:
    • 75µl DNA Digestion buffer x # of samples
    • 5µl DNase I x # of samples
  27. Added 80µl of DNase I treatment master mix directly to the filter of the spin column
  28. Began warming Nuclease-free H20 in the large incubator to 70 degrees C
  29. Added 400µl of RNA Pre-Wash buffer to each column and centrifuged at 16,000 rcf for 30 seconds
  30. Repeated step 29
  31. Added 700µl of RNA Wash Buffer and centrifuged at 16,000 rcf for two minutes
  32. Transferred spin columns to new 1.5mL tubes (final tubes)
  33. Added 50µl of warmed Nuclease-free H20 directly to the filter of the spin column and incubated at room temp for 5 minutes
  34. Centrifuged at 16,000 rcf for 30 seconds
  35. Repeated addition of H20 and spin
  36. Aliquoted 5µl of RNA for Qubit and TapeStation analysis in strip tubes
  37. Labeled 1.5mL tubes stored in -80

Qubit

Sample # Standard 1 (RFU) Standard 2 (RFU) RNA 1 (ng/µl) RNA 2 (ng/µl) Average RNA (ng/µl)
Mytilus Live 9 447 11422 256 258 257
Mytilus Live 10 447 11422 199 199 199
Mytilus Live 11 447 11422 312 314 313
Mytilus Live 12 447 11422 420 424 422

TapeStation

Results:

tape1 tape2 tape3 tape4

Written on March 12, 2019