RNA Isolation – Jessica’s Geoduck Larval Stages

Isolated RNA from the following samples provided by Jessica Blanchette (stored in RNA later):

  • Trocophore 1 (T1)
  • Trocophore 2 (T2)
  • Veliger 1 (V1)
  • Veliger 2 (V2)
  • Settlers Interphase 1 (S1)
  • Settlers Interphase 2 (S2)

The tocophore and veliger larval stages are neutrally bouyant (i.e. will not pellet when centrifuged). In order to separate them from the RNA Later, I used a fine mesh (don’t know mesh size; bag was labeled “Unknown”) as a “guard” between the pipette tip and the larvae. Removed RNA Later from those two groups in this fashion. However, a significant portion of the larvae in these tubes adhered to the outside of the mesh. I left the mesh “guard” in the tube, added 1mL of TriReagent and vortexed. The mesh quickly dissolved in the TriReagent, creating a milky white mix.

For the settlers samples, there was a such a large pellet already in the existing tubes, I just took ~75uL of this material, transferred to a clean tube and added 1mL of TriReagent. However, most of the debris that I transferred dissolved extremely quickly. I was expecting there to more insoluble “debris”, because marine bivalve larval shells generally don’t readily dissolve in the presence of TriReagent. So, I suspect that much of the settlers samples is not really geoduck larvae.

Due to time constraints, stored all samples O/N @-80C in TriReagent.

Samples were thawed and RNA was isolated, and DNased, using the Direct-zol RNA Miniprep Kit (ZymoResearch), eluted with 50uL of 0.1% DEPC-treated H2O, and spec’d on the NanoDrop1000.

Prior to isolation, sample V1 showed a clear phase separation that none of the other samples exhibited. Sample V1 had a pink, goopy layer on top of a clear, low-viscosity layer. All other samples retained the uniform pink coloration imparted by the TriReagent. Additionally, after addition of the EtOH in the procedure to sample V1, a large amount of white precipitate formed and settled to the bottom of the tube. This did not happen in any other samples.

Samples were stored @ -80C in “Shellfish RNA Box #5

Results:

Overall, the yields are relatively low, as expected. Virtually all of the samples have poor OD260/280 values. Although not shown, there was a consistent shift in peak absorbance from 260nm towards 270nm, leading to the poor OD260/280 values.

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