INTRO

I was asked to isolate RNA from a list of samples (GitHub Issue) from a number of Crassostrea gigas (Pacific oyster) spat and seed that had been collected as part of the project-gigas-carryover (GitHub repo).

MATERIALS & METHODS

Oysters had been previously preserved in RNAlater (Ambion), supernatant removed, and then frozen @ -80^oC. All samples were treated in the following fashion (12 spat were processed, followed by 12 seed samples.). Here’s the full list of samples (GitHub; commit 82f1a4b).

RNA Isolation

RNA isolations were conducted using the Directzol RNA Miniprep Kit (ZymoResearch).

Added 350uL of TriReagent (ZymoResearch) to existing 1.5mL tubes containing the sample.
Homogenized using disposble pestles.
Centrigured at 21,000g to pellet insoluble material.
Transferred 325uL of homogenate to a 1.5mL tube.
Added 325uL (i.e. equal volume) of 100% ethanol to homogenate and vortexed.
Transferred solution to spin columns.
Followed kit protocol, including DNase step.
Elutions:
- Spat: 50uL of H₂.
- Seed: 100uL of H₂.

Samples were temporarily stored on ice for quantification and reverse transcription.

RNA Quantification

RNA was quantified with the Roberts Lab Qubit 3.0 fluorometer (Invitrogen). Samples were quantified with either the Qubit RNA Broad Range (BR) asssay (Invitrogen) or, if a sample had insufficient concentrations of RNA to be detected, with the High Sensitivity (HS) assay (Invitrogen). 1uL of sample was used for the assay(s).

RESULTS

Raw Qubit data is here:

20240319-cgig-qubit-carryover-spat-seed.csv

The following spat samples didn’t yield any RNA:

The following seed samples didn’t yield any RNA:

Warning

The seed samples consisted of large quantities of tissue, which I think caused issues with the TriReagent extraction. This resulted in some strange results after homogenization and centfiguation. Many of them appeared to separate into phases (a dark red top phase and a clear bottom phase). Additionally, after combining with ethanol, there were large quantities of precipitate in most of the samples; these precipitates were not transferred to columns. The red phase still contained a lot of insoluble material, which possibly clogged columns and inhibited RNA binding.

Overall, it would seem that going forward, a significantly large quantity of TriReagent should be used and/or taking a section of the sample (instead of the entire sample) might be warranted. In regards to the latter, I’m not sure if this is desirable, as these are whole oysters and I’m not certain what the implications for comparative transcriptomics would be if using only a portion of the oyster.

Next up, create some cDNA…