Ronit’s Notebook: RNA Extraction for C.Gigas Desiccation + Elevated Temperature Samples (Round 2)

I decided to run another RNA isolation today with 8 more samples: D03, D04, D13, D14, T03, T04, T13, T14

Protocol was as follows:

1. 500 µL of RNAzol RT was added to a clean tube.
2. Tissue samples were removed and a small section was cut out for RNA extraction.
3. Tissue portions were placed in the tube and an additional 500 µL of RNAzol RT was added to bring the volume up to 1mL.
4. The samples were vortexed vigorously for 10 seconds
5. Samples were incubated at room temperature for 5 minutes.
6. 400 µL of DEPC-water was added to the samples.
7. Samples were centrifuged for 15 minutes at 12,000 g.
8. 750  µL of the supernatant was transferred to a new, clean tube and an equal volume of isopropanol was added to the sample.
9. The samples were vortexed vigorously for 10 seconds.
10. Samples were incubated at room temperature for 5 minutes.
11. Samples were centrifuged for 15 minutes at 12,000 g.

Due to time constraints, I decided to finish up the extraction later/quantify RNA using the Qubit and stored the RNA pellet suspended in isopropanol in the -80 freezer.

Ronit’s Notebook: Candidate Genes for qPCR

I’m interested in doing a hypoosmotic stress exposure after finishing up the qPCR for the desiccation + elevated temp. samples. However, if we’re going to compare gene expression between desiccation and hypoosmotic stress samples, it’s important that some genes linked to hypoosmotic stress are examined in the desiccation samples as well to provide a basis for comparison. Here are a few classes of genes that I think might be relevant to examine:

• Ion and amino acid channels 
  • LTrpC-8: mediates permeation for cations such as sodium, potassium, calcium
  • KCTD1: cysteine-rich protein, binds to KV channels
• Immune response
  • CARM1: Transfer of methyl groups to histone 3 for chromatin remodeling
  • H2AV: One of the 5 main histone proteins involved in the structure of chromatin
• Apoptosis genes 
• Calcium binding genes      

Ronit’s Notebook: Desiccation/Elevated Temperature Samples qPCR (HSP90, EF1)

Today, I ran a qPCR assay with the cDNA from the desiccation + elevated temperature samples. I examined heat shock protein (HSP90) and elongation factor (EF1, normalization gene) and ran 2 duplicates for each primer. I created a mastermix with 20 µL of forward primer, 20 µL of reverse primer, 400 µL of 2x qPCR master mix, and 320 µL of DEPC-treated water. 19 µL of the mastermix was put into each well and a subsequent 1 µL of cDNA was put in for each sample (water for the negative controls and gDNA for the positive controls).

Sam confirmed that the elongation factor primer works, so we should hopefully see amplification for the EF1 primer. I’ll be in next week to check up on the data/run some more RNA extractions.

Plate map
The wells are organized by rows and are in the order of: D01, D02, D09, D10, D11, D12, D19, D20, T01, T02, T09, T10, T11, T12, T19, T20 (samples cover 2 rows).

A1-A12 and B1-B4 are the EF1 replicate 1 samples.
C1-C12 and D1-D4 are the EF1 replicate 2 samples.
E1-E12 and F1-F4 are the HSP90 replicate 1 samples.
G1-G12 and H1-H4 are the HSP90 replicate 2 samples.
H5 is the no template control for EF1. H6 is the control with gDNA for EF1. and H7 is the replicate no template control for EF1. H8 is the replicate control with gDNA for EF1.
H9 is the no template control for HSP90. H10 is the control with gDNA for HSP90. H11 is the replicate no template control for HSP90. H12 is the replicate control with gDNA for HSP90.

Ronit’s Notebook: RNA Extraction Day 2

I finished RNA extraction protocol from the C. Gigas heat samples this week. Last week, I had extracted the RNA and was left with an RNA pellet, but I still had to complete the ethanol washes, so this week I finished the ethanol purification step and went to quantify the extracted RNA using the Qubit. The methodology I followed is described below:

RNA Extraction Wrap-Up: 

1. Stored RNA pellets (suspended in ethanol) were taken out from the -80 freezer and left to thaw for around 10 minutes.
2. Supernatant was removed from all samples and 400 μL of 75% ethanol was subsequently added to each sample.
3. Each sample was then centrifuged for 5 minutes at 1200 g.
4. Supernatant was once again removed from each sample and each sample was then microcentrifuged for approximately 10 seconds so that any residual ethanol could be removed.
5. 50 μL of DEPC water was added to each sample and samples were then vortexed to dissolve the RNA pellet. One sample’s (D11) RNA pellet did not fully dissolve, so an additional 50 μL of DEPC water was added and pellet was manually broken up by vigorously pipetting.

RNA Quantification (Qubit)

1. 3980 μL of Qubit buffer and 20 μL of Qubit dye were added to a tube to create a 200:1 ratio between buffer and dye.
2. 198 μL of the mastermix and 2 μL of the RNA samples were added to each of 16 Qubit tubes (1 tube for each sample).
3. 2 standardization tubes were also set up. 190 μL of mastermix and 10 μL of Qubit standard #1/2 were added to 2 Qubit tubes.
4. Qubit assay was run, but RNA content in samples was beyond the limit of quantification for the machine. I will have to dilute the RNA samples and then re-quantify on Friday which should hopefully solve the problem.

 

Ronit’s Notebook: RNA Extraction for C.Gigas Heat Stress Samples

I started working with the C. Gigas heat stress samples to extract RNA for gene expression analysis. Sam took care of 8 of the samples and I did the RNA extraction for the other 8 samples: D09, D10, D11, D12, T09, T10, T11, T12

The protocol I followed is as follows:

1. 500 µL of RNAzol RT was added to a clean tube.
2. Tissue samples were removed and a small section was cut out for RNA extraction.
3. Tissue portions were placed in the tube and an additional 500 µL of RNAzol RT was added to bring the volume up to 1mL.
4. The samples were vortexed vigorously for 10 seconds
5. Samples were incubated at room temperature for 5 minutes.
6. 400 µL of DEPC-water was added to the samples.
7. Samples were centrifuged for 15 minutes at 12,000 g.
8. 750  µL of the supernatant was transferred to a new, clean tube and an equal volume of isopropanol was added to the sample.
9. The samples were vortexed vigorously for 10 seconds.
10. Samples were incubated at room temperature for 5 minutes.
11. Samples were centrifuged for 15 minutes at 12,000 g.
12. The supernatant was discarded and 400 µL of 75% ethanol was added to the samples.
13. Samples were centrifuged for 1.5 minutes at 4,000 g.
14. Supernatant was discarded and an additional 400 µL of 75% ethanol was added to the samples.
15. Samples were stored in -80 degree freezer.

Due to time constraints, we decided to finish up the extraction next week/quantify RNA using the Qubit and stored the RNA pellet suspended in ethanol in the -80 freezer.

 

Ronit’s Notebook: qPCR Prep/Spreadsheet Organization

In preparation for the RNA extraction from the heat stress C. Gigas samples, Sam and I went over some sample organization/prep. I updated my spreadsheet, which was linked in my previous notebook entry, so that it now has columns to show treatment, ploidy, and tissue type. Also–there are now entries for replicate samples as well (i.e. D01-C2), so that every sample tube has a corresponding entry in the spreadsheet.

I then chose 2 samples from each treatment to prep for RNA extraction and subsequent qPCR. As there were 8 distinct treatment groups, we pulled a total of 16 samples:

D01, D02 — Diploid oysters exposed to control conditions (water in aquarium)

D09, D10 — Diploid oysters exposed to control conditions (water in aquarium); subsequently exposed to 1 hr acute heat shock at 45 degrees Celsius.

D11, D12 — Diploid oysters exposed to desiccation + elevated temperature (27 degrees Celsius) for 24 hrs

D19, D20 — Diploid oysters exposed to desiccation + elevated temperature (27 degrees Celsius) for 24 hrs; subsequently exposed to 1 hr acute heat shock at 45 degrees Celsius.

T01, T02 — Triploid oysters exposed to control conditions (water in aquarium)

T09, T10 — Triploid oysters exposed to control conditions (water in aquarium); subsequently exposed to 1 hr acute heat shock at 45 degrees Celsius.

T11, T12 — Triploid oysters exposed to desiccation + elevated temperature (27 degrees Celsius) for 24 hrs

T19, T20 — Triploid oysters exposed to desiccation + elevated temperature (27 degrees Celsius) for 24 hrs; subsequently exposed to 1 hr acute heat shock at 45 degrees Celsius.

These tubes were pulled from the -80 freezer and kept in a new, unlabeled box (which is also in the -80 freezer and has been updated in the -80 Inventory Map). We then made sure that we had all the materials on hand for the RNA extraction next week.

Ronit’s Notebook: qPCR Trial Run Data

Today Sam and I took a look at the qPCR data from last week’s trial run. We found that the third primer we used (CYP1A) is not functional, as there was no amplification in wells that had the CYP1A primer. Elongation factor was supposed to be our normalizing gene target, but we found there to be a relatively high range in Cq values for the elongation factor gene across treatments (23.92–32.97). This may be due to an outlier cell (1H) which could have been caused by a pipetting error or sample contamination. For future qPCR runs, we will include technical replicates to increase the reliability of the data (we decided not to use technical replicates for this round as it was only a practice run).

Curves for the HSP90 primers looked good and melt curves for elongation factor and HSP90 indicated that there was only one amplification product in each well. We also reviewed some of the basics of qPCR, including how the curve threshold is determined, melt curves, etc.

Link to qPCR data: http://owl.fish.washington.edu/scaphapoda/ronit/admin_2018-09-12%2015-40-46_BR006896.pcrd

Notes about well setup:

1A–1D: Control treatment, elongation factor primer

1E–1H: Row 8 Column 1: CO2 treatment, elongation factor primer

2A–2D: Control treatment, HSP90 primer

2E–2H: CO2 treatment, HSP90 primer

3A–3D: Control treatment, CYP1A primer

3E–3H: CO2 treatment, CYP1A primer

4A–4C: Negative controls (no cDNA)

Ronit’s Notebook: qPCR Assay Trial Run

Today, I worked with Sam to do a trial qPCR assay using some old juvenile oyster samples from a graduate student’s experiment. We used 8 cDNA samples (4 control, 4 CO2 exposed) and looked at 3 different genes: elongation factor, HSP90, and CYP1A. Sam showed me the calculations for creating a 15 microliter master mix and I created master mixes for the 3 gene targets. For each master mix we added: 5′ primer, 3′ primer, qPCR master mix, and water. I’ve attached a picture of our calculations below:

We decided to multiply each volume by 8.8 (10% greater than the actual needed amount) in order to ensure that we had enough master mix. I then transferred 15 microliter of each master mix to the well plate along with 5 microliter of cDNA sample. 3 negative control samples were also kept on the plate in which I put 5 microliter water instead of cDNA. Note: we initially forgot to account for the negative control samples in our master mix calculations, so we had to go back and add additional master mix volume to the original solution (which is what the red numbers on the right indicate).

We then took the well plates to the qPCR machine where Sam walked me through the protocol for the amplification phase as well as the theory behind it. We left the samples running (the machine stated that it would take approximately 1hr 30 minutes to complete) and will check the curves when I come in to the lab next.

Samples were kept on top of the box labelled 1 in the 2nd freezer:

Ronit’s Notebook: Link to Spreadsheet and Labelling System Notes

Attached below is a link to the Excel spreadsheet for the C. Gigas exposure from 8/28-8/29. The column on the left indicates the oyster’s label (see labelling system below) and the subsequent column records the weight of the oyster. All oysters were weighed prior to shucking on 8/29.

Spreadsheet: https://docs.google.com/spreadsheets/d/e/2PACX-1vTHPB2YUKnaZ_-OKJR2fWATWSlcDFADULn_1pjz1_2BYh9gSpRW-VHKwpdCFmlvxfiR1C8g7DsAoxHx/pubhtml?gid=0&single=true

Labelling system: 

D01-D08: Diploid oysters exposed to control conditions (water in aquarium)

D09-D10: Diploid oysters exposed to control conditions (water in aquarium); subsequently exposed to 1 hr acute heat shock at 45 degrees Celsius.

D11-D18: Diploid oysters exposed to desiccation + elevated temperature (27 degrees Celsius) for 24 hrs

D19-D20: Diploid oysters exposed to desiccation + elevated temperature (27 degrees Celsius) for 24 hrs; subsequently exposed to 1 hr acute heat shock at 45 degrees Celsius.

T01-T08: Triploid oysters exposed to control conditions (water in aquarium)

T09-T10: Triploid oysters exposed to control conditions (water in aquarium); subsequently exposed to 1 hr acute heat shock at 45 degrees Celsius.

T11-T18: Triploid oysters exposed to desiccation + elevated temperature (27 degrees Celsius) for 24 hrs

T19-T20: Triploid oysters exposed to desiccation + elevated temperature (27 degrees Celsius) for 24 hrs; subsequently exposed to 1 hr acute heat shock at 45 degrees Celsius.

Ronit’s Notebook: Desiccation + Elevated Temperature Exposure Set-up Day 1

Yesterday (8/28), we set up a 24 hr exposure with triploid and diploid C. Gigas oysters to an elevated temperature + desiccation stress. In total, we had 40 oysters (20 diploid, 20 triploid). 10 control animals were kept for both the diploid and triploid animals in mesh bags in the aquarium in regular seawater. We had to place a foam cover over the mesh so that sea stars wouldn’t get in and potentially interfere with the controls. The other 20 animals (10 diploid, 10 triploid) were transferred to an incubator at 27 degrees Celsius  where they were also kept in mesh bags. Exposure began at roughly 12:00 PM. Additionally, we decided to take 2 animals from both the control and the desiccation + elevated temperature exposure at 24 hrs to expose to a 1 hr acute heat shock at 45 degrees Celsius (for a total of 8 oysters exposed to acute heat shock). Thus, our sample size is N = 8 for the diploid/triploid controls and the diploid/triploid desiccation + elevated temperature treatment group.

Originally, we were planning to do a factorial stress response experiment, examining the effects of hypoosmotic stress (low salinity), desiccation + elevated temperature, and the two stressors combined. However, we ultimately decided to go with a simpler experimental design that would be more robust and could give us a baseline stress response to work with. I’m still interested in investigating interactive effects between multiple stressors, especially in triploids, so once we have this baseline single-stressor trial completed, we can definitely think about doing a factorial design!

Today (8/29), we’ll be sampling the oysters at the 24 hr mark, collecting mantle, adductor muscle, and gill tissue for follow-up RT-qPCR. At around noon, we’ll take 2 oysters each from the diploid/triploid control and treated groups to do an acute heat shock at 45 degrees Celsius for 1 hour.