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.

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.