Today I tried the TrizolLS Reagent extraction again, but added a vortex (5s) step after the addition of chloroform to the samples. Throughout the extraction, the samples looked pretty similar to how they did during my last attempt, and the Qubit results were the same: “out of range”.

Here’s the protocol of what I did today:

Samples used:

From Day 26:

Trizol LS Reagent Extraction Protocol:

Lyse samples and separate phases:

  1. Add 750uL Trizol LS Reagent to the samples (pelleted C. bairdi hemolymph)
    (Need to maintain a 3:1 ratio of Trizol to sample. I estimated that the samples are about 250ul)
  2. Homogenize sample by pipetting (5x per sample)
  3. Centrifuge 5 mins at 12000g at 4C
  4. Transfer clear supernatant to a new, labeled tube (The pink Trizol should be at the bottom, but in both attempts, the Trizol was on top, so I had to pipet it out to get to the clear “super”-natant)
  5. Incubate 5 mins to permit complete dissociation of the nucleoproteins complex.
  6. Add 200ul of chloroform and close lid
  7. Vortex 5s
  8. Incubate at room T 2-3 mins
  9. Centrifuge 15mins at 12000g at 4C
    (The mixture separates into a lower phenol-chloroform, and interphase, and a colorless upper aqueous phase)
  10. Transfer aqueous phase containing RNA to a new tube by angling at 45˚ and pipetting out.
    (DO NOT transfer any of inerphase or organic layer. ONLY the aqueous phase)

Isolate RNA

A. Precipitate RNA

  1. Add 500ul of ispropanol to the aqueous phase
  2. Incubate at room T 10 mins
  3. Centrifuge 10 mins at 12000g at 4C
    (Total RNA becomes a white gel-like pellet at the bottom of the tube. In both attempts, it was hard to see. It was never white - always clear at the bottom, so I just continued with the protocol as though there were a gel-like pellet at the bottom.)
  4. Discard supernatant

B. Wash the RNA

  1. Resuspend pellet in 1mL of 75% ethanol
  2. Vortex briefly. Centrifuge for 5 mins at 7500g at 4C
  3. Discard supernatant
  4. Air dry pellet 10 mins (This part was challenging in both attempts because I couldn’t see any pellet at the bottom.)

C. Solubilize the RNA

  1. Resuspend pellet in 20ul of 0.1% DEPC-treated H20
  2. Incubate in a heat block set to 55˚C for 15mins

Run 1ul of each sample on Qubit

Results: All three tubes were “out of range”

Next steps thoughts:

  • Go back and try the Qiagen Kit again
  • Maybe try the kit or Trizol LS with other hemolymph samples from other animals? Or maybe tissue samples from other animals? Just to see if the process works? (A thought that Shelly had while we were talking today)