Ronit’s Notebook: Identifying Unknown Oyster Sample from Marinelli Shellfish Company (C. gigas vs C. sikamea)

The Marinelli Shellfish Company had an issue with one of their bags of oysters being labelled as Kumamoto oysters (C. sikamea) AND as Pacific Oysters (C. gigas). Obviously, confusion abounded, and ultimately we were tasked with figuring out what the true identity of these mystery oysters were. To do so, DNA was isolated from mantle tissue from the unknown oysters, a sample set of known C. gigas, and a sample set of known C. sikamea. 4 PCR primers were targetting the cytochrome oxidase gene: universal forward and reverse primers (HC02198, LCO1490); reverse primer specific to C. gigas (COCgi269r); and a reverse primer specific to C. sikamea (COCsi546r). Note: this was a multiplex PCR.

Cycling parameters were as follows:

95°C for 10 mins; 30 cycles of 95°C (1 min), 51°C (1 min), 72°C (1 min); 72°C (10 min).

PCR reactions were run on a gel and results are visualized below:

The first set of 4 samples (offset by ladders) are the unknown samples; the second set of 4 samples are C. gigas; and the third set of 4 samples are C. sikamea.

Using the GeneRuler DNA Ladder as a guide,

First, we can see that there is a band of approximately 700bp in all samples, indicating that the universal forward and reverse primers did their job (positive primer). Next, we expect to see a band of approximately 260-270 bp in the known C. gigas samples, which we do! Similarly, we also expect to see a band of 550 bp in the known C. sikamea samples, which we also do. (Note: it looks like there is a faint band of 270bp in the C. sikamea samples. Could be a sign of contamination with C. gigas samples?).

In the unknown samples, a prominent band of 270bp is clearly visible, which is what we should see in C. gigas samples, Thus, it seems that these mystery samples are in fact C. gigas. Case closed!

Ronit’s Notebook: Finalized qPCR Plots

I worked on figuring out some of the stats for my qPCR data with Shelly on Thursday. I decided to go with an ANOVA/Tukey’s Honest Significance Difference Test, with the data being normalized using a log transform. Final qPCR plots are attached below–capital letters indicate differences in treatment, asterisks denote differences between individual groups, and capital letters next to ploidy key indicate differences between diploids and triploids.

Ronit’s Notebook: Adjusted qPCR Data, COX1 Relative Mitochondrial Abundance Plot

To account for N/A Cq values, I substituted in a Cq value of 45 wherever the Cq value was nonexistent. This allows for some initial analysis of the data to see which genes might warrant further work. Below are the adjusted qPCR plots. I also generated the COX1 Cq plot (not normalized to actin) to examine relative mitochondrial abundance between stressed and non-stressed diploids and triploids.

Ronit’s Notebook: qPCRs and Data Analysis

I ran SOD and ATP Synthetase qPCRs last week. Currently, I’m working on getting box plots worked out with the R script–the main issue I’m trying to sort out is how to account for the N/A values for some of the samples as the current plots just ignore it. I’m also adding a statistical analysis portion to the script so that we can start visualizing which treatments are statistically different.

Link to ATP Synthetase qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2019-01-23%2014-56-33_BR006896.pcrd

Link to SOD qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2019-01-09%2015-08-24_BR006896.pcrd

Ronit’s Notebook: Generating Plots for qPCR Data

Today, I made a GitHub account and generated box plots for all my qPCR data using R. Attached below are pictures of the box plots:

Ronit’s Notebook: HSC70, MBD2, and MeCP2 qPCR

I ran a qPCR assay on my desiccated + elevated temp. samples with HSC70, MBD2, and MeCP2 as my gene targets. For protocol used, please refer to previous notebook entries.

Link to Excel spreadsheet of Cq values: https://docs.google.com/spreadsheets/d/1AhdJLEcXt6vnNBcGVLnc926KlZ5J-Nt8JMpiCdrUvaU/edit?usp=sharing

Link to HSC70 qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2018-12-19%2016-24-55_BR006896.pcrd

Link to MBD2 qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2018-12-20%2013-17-34_BR006896.pcrd

Link to MeCP2 qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2018-12-20%2014-58-17_BR006896.pcrd

Ronit’s Notebook: DNMT1 and Actin qPCR

In the last two qPCR assays, no fluorescence was detected. Turns out we were using Promega master mix which is specifically for probe-based assays…as such, it doesn’t have any fluorescent dye in it, explaining the lack of fluorescence detection in any of the assays. I switched over to using a different master mix that should solve the issue.

I reran the DNMT1 primer assays and will run an actin qPCR to determine if it might be a possible normalizing gene. For protocol used, please refer to previous lab book entries.

Link to Excel spreadsheet of Cq values for both actin and DNMT1: https://docs.google.com/spreadsheets/d/1AhdJLEcXt6vnNBcGVLnc926KlZ5J-Nt8JMpiCdrUvaU/edit?usp=sharing

Link to DNMT1 qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2018-12-12%2015-24-28_BR006896.pcrd

Link to actin qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2018-12-14%2016-16-16_BR006896.pcrd

Plate map:
All replicates are in adjacent wells. The order of the samples in the well plate by rows is: D01, D02, D03, D04, D05, D06, D07, D08, D09, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, T01, T02, T03, T04, T05, T06, T07, T08, T09, T10, T11, T12, T13, T14, T15, T16, T17, T18, T19, T20

Example:
Well—–Sample
A01——D01
A02——D01
A03——D02
A04——D02

Ronit’s Notebook: qPCR with Desiccation + Elevated Temp. Samples (DNMT1)

Before the long weekend, I ran a qPCR assay with my desiccated + elevated temp. samples. There were 40 samples in total and I ran one plate with DNMT1 (DNA methyltransferase) as my gene target.

Plate Layout:

The plate is laid out with duplicates in adjacent wells and the order of samples is as follows:

D01, D02, D03, D04, D05, D06, D07, D08, D09, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, T01, T02, T03, T04, T05, T06, T07, T08, T09, T10, T11, T12, T13, T14, T15, T16, T17, T18, T19, T20

There are 2 positive controls with gDNA in wells G9 and G10. There is a no-template control (negative control) with no template in wells G11 and G12.

To run the qPCR assay, 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). Note that all sample cDNA used was a 1:5 dilution.

RNA Extraction Wrap-Up for Desiccation + Elevated Temp. Samples (11/8)

On 11/8, I finished up the RNA extraction for the final 16 samples of the desiccation + elevated temp. exposure (D05, D06, D07, D08, D15, D16, D17, D18, T05, T06, T07, T08, T15, T17, T18). I also ran the Qubit assay for 24 samples (D03, D04, D05, D06, D07, D08, D13, D14, D15, D16, D17, D18, T03, T04, T05, T06, T07, T08, T13, T14, T15, T16, T17, T18). 4 samples (D06, T05, T15, T17) had RNA concentrations above the limit of quantification, so I will have to dilute those samples and re-run the Qubit assay. Described below is the protocol for both the RNA extraction wrap-up and the Qubit assay:

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.

 

Ronit’s Notebook: RNA Extraction for Remaining C. Gigas Dessication + Elevated Temperature Samples

I extracted RNA for 16 samples (D05, D06, D07, D08, D15, D16, D17, D18, T05, T06, T07, T08, T15, T16, T17, T18). RNA pellets were stored in the -80 freezer following isopropanol precipitation. The protocol I used is described below:

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.

Note: one of the RNA pellets (D17) looked black. Not sure what could have caused this or if this is a sign of contamination, but I’ll proceed with the RNA extraction for D17 and see what the Qubit results show for that sample.