RNA Isolation and Quantification - C.gigas Lifestage Carryover Seed Juvenile and Adult

2024
project-lifestage-carryover
Crassostrea gigas
Pacific oyster
seed
adult
juvenile
RNA HS assay
Qubit
RNA isolation
Directzol RNA Miniprep
Author

Sam White

Published

June 26, 2024

INTRO

I was asked to isolate RNA from a list of samples (GitHub Issue) from a number of Crassostrea gigas (Pacific oyster) spat, seed, and adults that had been collected as part of the project-gigas-carryover (GitHub repo). The full list of all samples needing processing is here:

I processed the following eight samples:

  • 280
  • 287
  • 290
  • 291
  • 293
  • 294
  • 295
  • 297

MATERIALS & METHODS

Oysters had been previously preserved in RNAlater (Ambion), supernatant removed, and then frozen @ -80oC. All samples were treated in the following fashion:

RNA Isolation

Samples were thawed and residual RNAlater (Ambion) was removed.

RNA isolations were conducted using the Directzol RNA Miniprep Kit (ZymoResearch). All centrifugation steps were performed at 16,000g in an Eppendorf 5425 microfuge at room temperature, unless noted otherwise.

  • Added 500uL of TriReagent (ZymoResearch) to existing 1.5mL tubes containing the sample.
  • Homogenized using disposble pestles.
  • Transferred to a 15mL conical.
  • Adjusted volume in 0.5mL incremements until TrReagent was no longer cloudy.
  • Pelleted insoluble material 10,000g for 5mins in SL50T rotor (Sorval) in Super T21 table top centrifuge (Sorval) at room temp.
  • Transferred supe to new 15mL conical containing equal volume of 100% ethanol and vortexed.
  • Transferred mixture to spin columns in 700uL volumes.
  • Followed kit protocol, including DNase step.
  • Eluted in 50uL of H2O.

Samples were temporarily stored on ice for quantification. DNAsed RNA samples were subsequently transferred to the same box in the -80oC from which they were taken.

RNA Quantification

RNA was quantified with the Roberts Lab Qubit 3.0 fluorometer (Invitrogen). Samples were quantified with the Qubit RNA High Sensitivity (HS) assay (Invitrogen). 1uL of sample was used for the assay(s).

Note

Samples 280 and 293 were initially too concentrated. Sample 293 was diluted with an additional 50uL of H2O and measured again. Sample 280 required two rounds of 50uL dilutions before it fell into the assay range.

RESULTS

Raw Qubit data is here:

sample_ID Original sample conc. (ng/uL)
287 15.5
290 47
291 39.2
294 180
295 196
297 156
293 140
280 150

SUMMARY

All samples yielded RNA.