Looking at the Oly data from the 2017 Oly project, I see a couple patterns. Check out the below image, where I simply identify the treatment within each population that had the MOST and LEAST of the following:
- Gonad ripeness post-OA
- Larval production
- % larval survival
The coding as as follows:
- 6L: 6degC, low pH
- 6A: 6degC, ambient pH
- 10L: 10degC, low pH
- 10A: 10degC, ambient pH
- The group that spawned the most number of larvae (normalized per oyster) consistently had the poorest larval survival across all populations.
- The South Sound F1 and F2 groups have identical patterns, indicating a genetic component in how the environment impacted phenotype.
- Some similar patterns between Hood Canal and Fidalgo Bay, but less so than the SS F1 and F2
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In a previous post I generated pie charts of the 2017 Oly gonad stage and sex. Here, I run some quick stats on the gonad stage and sex data to confirm that my visually determined differences in maturation between 6degC low pH vs. 6degC ambient pH is, indeed, statistically different. I performed these analyses in R in my Histology-Pie-Charts.R script.
I created contingency tables to analyze differences in Sex and Stage, then ran chi-squared or Fisher’s exact tests, depending on sample size (small n = Fisher’s).
Contingency Table – All populations, by Sex
A warning was printed along with these results… “Warning message: In chisq.test(CT.Sex) : Chi-squared approximation may be incorrect”. So, I ran a Fisher’s Exact Test for Count Data instead of a chi-squared test:
Contingency Table – All populations, by Stage; Stages are significantly different between treatments, and indicate that low pH treatment resulted in more early-stage gonad.
Let’s dial down into each poplulation. Bear with me here, there are a ton of contingency tables and stats results. Note I ran Fisher’s test for all these due to sample size:
Tables, by SEX
Tables, by STAGE:
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Using PAXgene Tissue DNA Kit (see Kit Handbook) on 2017 Oly gonad tissue fixed in paraffin wax. Tissue was fixed using the PAXgene fixative/stabilizer kit.
Things that I need, in addition to the kit:
• Xylene (we have Xylene Cyanol FF in the chemical storage). NEED XYLENE.
• Homogenizer NEED HOMOGENIZOR. question; do I need to be worried about shearing the DNA? PAXgene advises to “optimize” the homogenization time to not fragment DNA, but that’s one of the next steps that I will do in the workflow…
• Scalpels • Ethanol (96–100%, purity grade p.a.)
• Pipets* (10 µl – 1 ml)
• Sterile, aerosol-barrier, RNase-free pipet tips
• Variable-speed microcentrifuge* capable of attaining 1000–20,000 x g, and
equipped with a rotor for 2 ml microcentrifuge tubes
• Shaker-incubator* capable of incubating at 56°C, 70°C, and 80°C, and shaking at ≥400 rpm, not exceeding 1400 rpm (e.g., Eppendorf® Thermomixer Compact, or equivalent) • Vortex mixer*
• Crushed ice
• Optional: RNase A (100 mg/ml; cat. no. 19101)
The handbook provides instructions for DNA-only or RNA/DNA extraction combined. Do I want RNA in my samples?:
The PAXgene Tissue DNA procedure copurifies DNA and RNA when both are present in the sample. Transcriptionally active tissues such as liver and kidney contain high levels of RNA, which will be copurified. RNA may inhibit some downstream enzymatic reactions, although it does not affect PCR. If RNA-free genomic DNA is required, RNase A should be added to the sample before addition of Buffer TD2, to digest the RNA.
Protocol requires Xylene, which I will purchase unless I hear differently. (Note: we have Xylene Cyanol FF, but I believe that is for staining purposes, not a solvent).
Following Sam and Yaamini’s notebooks, I’ll need Qubit™ dsDNA HS Assay Kit for quantification; I don’t see this in inventory, but are there enough supplies left in the kit for 20 samples?
Submitted issue to GitHub with the above questions. Probably won’t be able to actually execute lab work tomorrow after all…
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