Len's+Notebook

Date November 29, 2011

1. The expression of metallothionein gene between diploid and triploid oysters in low pH (5.24) conditions was relatively similar. This result could be attributed to the fact that a low pH environment did not elicit an upregulation of the metallothionein gene (user:emmatsIt could also be that the oysters were dead and the RNA had degraded). As a result, both diploid and triploid oysters expressed a small, but sufficient and relatively equal, expression of metallothioneins independent of the acidic seawater. Instead, the expression could have possibly been in response to the small amount of xenobiotics in the environmental conditions. Another possibility is that the relatively similar, normalized values of gene expression for both groups was already upregulated to the similar degree. Given the same environmental conditions, the oysters could have upregulated their metallothionein gene expression from no gene expression to gene expression in response to the 5.24 pH seawater environment. Ploidy could not have played a significant role in the amount of gene expression, and that upregulation was only dependent on the acidic seawater.

2. One obstacle in terms of designing an experiment is creating one to accommodate such a large group. As such, the group decided to test different aspects of the oysters and their environment: diploid vs. triploid, low pH vs. high pH, wet vs. dry. Although eventually figured out, it was hard deciding and coordinating what aspects of the study each member wanted to focus on. Even more so, it was hard coordinating what tissues to sample and how much tissue to extract for a sufficient number of samples for each person. Another obstacle was efficiently extracting RNA from a high number of samples and storing them on ice as fast as possible to prevent degradation. When sampling such a high number of tissues, it's hard to effectively remove all the EtOH in the protein extraction phase to obtain clean RNA results. The first obstacle may have resulted in less, but still a sufficient amount (n=3), of oysters to obtain gene expression results. The second obstacle may have produced unpure RNA from EtOH and protein residues, but still produced decent results.

3. What could potentially be important about this research is to learn about oysters in terms of aquaculture and the natural environment. Increasing CO2 levels are without a doubt causing ocean acidification. Such an event poses a stress to marine organisms, one species being the oyster. If the results are as expected, then oysters would constantly be upregulating a package of stressor genes to deal with any stress response. This would cause physiological changes in the organism, perhaps diverting energy away from reproduction (user:emmatsand other physiological processes) to survival, which has implications for decreased progeny, size, and ultimately the population. Oysters are part of a dynamic food web, including those of humans, and the dwindling of oyster populations around the world suggests an impact on oyster predators and preys in its ecosystem. Also, with aquaculture, ocean acidification may disrupt the balance of the marine ecosystem and cause a huge drop in available seafood, causing an increase in prices for fresh fish, oysters, clams, etc. By understanding the stress response of marine organisms like the oyster, measures can be implemented to prevent the extinction or endangerment of marine organisms. Furthermore, MT levels responding to ocean acidification will allow the oyster to be a bioindicator for not only the level of xenobiotics in the environment, but as a means of determining relative stress/ocean acidification on marine organisms as well.

4. One part of my research that has completely stumped me is the exact impact of ocean acidification on marine organisms. Studying ocean acidification in oysters is a way of viewing acidification on a smaller scale level by viewing its effect on specifically oysters, but even understanding the results and interpreting them to a bigger picture is only an estimate of its potential impact. What I would like to know more is the exact impact of ocean acidification on marine organism relationships and its impact on humans in terms of social, economic, and even political aspects. There is a plethora of literature on ocean acidification that has been done, however its really only a phenomena that at this point, is still developing, and it remains uncertain the exact impact it will have on marine organisms as it continues to progress. If global measures aren't taken to reduce ocean acidification, I think the results will be revealed soon enough within this century.

5. Amiard-Triquet, C. "Use of Metallothionein in Gills from Oysters (crassostrea Gigas) As a Biomarker: Seasonal and Intersite Fluctuations." Biomarkers . 7.2 (2002). Print.
 * The study of this paper aims to support the idea of using Crassostrea Gigas gill tissue as a legitimate biomarker for assessing metal concentrations in the environment. The study looked at Japanese oysters in a low metal concentrated control site (Bay of Bourgneouf), Japanese oysters in a high metal concentrated site (Gironde estuary), and translocated oysters from Bourgneuf to Gironde. Their findings supported the idea of using oysters as a bioindicator due to the positive correlation between MT expression in their gills to levels of metals in the water.

Marie, V, P Gonzalez, M Baudrimont, I Boutet, D Moraga, JP Bourdineaud, and A Boudou. "Metallothionein Gene Expression and Protein Levels in Triploid and Diploid Oysters Crassostrea Gigas After Exposure to Cadmium and Zinc." Environmental Toxicology and Chemistry / Setac . 25.2 (2006): 412-8. Print.
 * The study of this paper aims to observe the differences in metallothionein gene expression in triploid and diploid Crassostrea Gigas after exposure to cadmium and zinc. The authors used qPCR as a means of assessing relative gene expression of the different isoforms of metallothionein (i.e. CgMT1, CgMT2, CgMT3) before and after exposing the oysters to Cd, Zn, and Cd+Zn. In their experiment, they found that relative gene expression between diploid and triploid oysters were similar, and that ploidy apparently does not play a role in MT gene expression.

Date November 21, 2011

__Summary__ cDNA samples of C. Gigas gill tissue in low pH were prepared to run in quantitative PCR. Also, the RNA extracted and used to obtain the cDNA samples was quantitated using a nanodrop.

__Materials__
 * Refer to qPCR PROTOCOL materials in November 14, 2011 lab
 * Refer to RNA QUANTIFICATION PROTOCOL materials in October 11, 2011 lab
 * C. Gigas low pH gill tissue cDNA samples (5x 3n, 2x 2n samples)
 * C. Gigas low pH + control RNA samples
 * Low pH: 4x 2n samples, 5x 3n samples
 * Control pH: 3x 3n dry, 3x 3n wet, 3x 2n dry, 3x 2n wet samples
 * Metallothionein Cg_MTIV_F + Cg_MTIV_R primers
 * Elongation Factor 1 Forward and Reverse primers
 * 5x 8-well qPCR clean wells + caps

__Methods__

qPCR PROTOCOL
 * 1) Refer to qPCR PROTOCOL in November 14, 2011 lab
 * 2) Make a master mix for 21 samples with metallothionein promoter forward and reverse. 18 samples + 2 controls + 1 extra
 * 3) This will be (in microliters): 275 2x immomix, 22 Syto, 27.5 upstream primer, 27.5 downstream primer, and 154 ultra pure water
 * 4) Make a master mix for 21 samples with elongation factor 1 promoter forward and reverse. 18 samples + 2 controls + 1 extra
 * 5) This will be (in microliters): 275 2x immomix, 22 Syto, 27.5 upstream primer, 27.5 downstream primer, and 154 ultra pure water
 * 6) Label the 5x 8-well qPCR wells - 20 wells for metallothionein (MT), 20 wells for elongation factor 1 (EF1)
 * 7) Each gene (MT + EF1) will have 5 triploid (3n) oyster cDNA and 4 diploid (2n) oyster cDNA. Two replicates of each sample per gene.
 * 8) Label specifically - i.e. 3n(1), 3n(2), 2n(1), 2n(2), neg. control
 * 9) Run in qPCR.

RNA QUANTIFICATION PROTOCOL
 * 1) Refer to RNA QUANTIFICATION PROTOCOL in October 11, 2011 lab
 * 2) Record the RNA concentration (nanogram/microliter), A260/280 ratio, and A260/230 ratio

__qPCR Results__ After inputting results from the qPCR into the PCR miner program, an average efficiency of genes for MT and EF1 were 0.771661 and 0.812465 respectively. In further analysis, the average CT values of the specific samples were inputted into the formula: 1/(1+AvgGeneEfficiency)^(Average CT). This number was then compared between MT and EF1 by dividing MT value/EF1 value to generate a quantified gene expression value (MT/EF1):


 * Table 1 Gene Expression for Triploid (3n) Metallothionein vs. Triploid (3n) EF1** **in low pH**
 * ||  ||   || MT_3N1 || MT_3N2 || MT_3N3 || MT_3N4 || MT_3N5 ||
 * Avg CT || 37.2117 || 37.3717 || 34.6253 || 37.6828 || 19.105 ||
 * Formula || 5.71919E-10 || 5.21908E-10 || 2.51042E-09 || 4.3684E-10 || 1.79758E-05 ||
 * || EF1_3N1 || EF1_3N2 || EF1_3N3 || EF1_3N4 || EF1_3N5 ||
 * Avg CT || 17.5069 || 17.0771 || 16.3869 || 17.3633 || 16.7609 ||
 * Formula || 3.00954E-05 || 3.88601E-05 || 5.85816E-05 || 3.27783E-05 || 4.68995E-05 ||
 * MT/EF1 || 1.90036E-05 || 1.34304E-05 || 4.28534E-05 || 1.33271E-05 || 0.38328302 ||  ||
 * MT/EF1 || 1.90036E-05 || 1.34304E-05 || 4.28534E-05 || 1.33271E-05 || 0.38328302 ||  ||
 * MT/EF1 || 1.90036E-05 || 1.34304E-05 || 4.28534E-05 || 1.33271E-05 || 0.38328302 ||  ||


 * || MT_2N1 || MT_2N2 || MT_2N3 ||  || MT_2N4 ||
 * Avg CT || 37.0664 || 35.8266 || 35.5372 ||  || 0 ||
 * Formula || 6.21476E-10 || 1.26289E-09 || 1.49021E-09 ||  || 1 ||
 * || EF1_2N1 || EF1_2N2 || EF1_2N3 ||  || EF1_2N4 ||
 * Avg CT || 16.5349 || 18.5034 || 17.0896 ||  || 19.1069 ||
 * Formula || 5.3646E-05 || 1.66393E-05 || 3.85723E-05 ||  || 1.16217E-05 ||
 * MT/EF1 || 1.15848E-05 || 7.58985E-05 || 3.86343E-05 ||  || 86046.25441 ||
 * Table 2 Gene Expression for Diploid (2n) Metallothionein vs. Diploid (2n) EF1 in low pH**
 * MT/EF1 || 1.15848E-05 || 7.58985E-05 || 3.86343E-05 ||  || 86046.25441 ||
 * Table 2 Gene Expression for Diploid (2n) Metallothionein vs. Diploid (2n) EF1 in low pH**

__qPCR Conclusion__ The average efficiency of the genes was decent. Ideally, it should be greater than 0.85, however the values obtained were good enough for further analysis. The average CT values obtained were inputted into the formula to quantify gene expression. Since EF1 was the control gene in the qPCR reaction, the MT numbers were normalized to the control EF1 to determine how much metallothionein gene was expressed. This is reflected in the MT/EF1 row of both table 1 and 2 above. As seen from the tables, the relative MT/EF1 value was less than one, indicating MT gene expression is much less than the normalized EF1 gene expression in both diploids and triploids. When comparing between triploids and diploids, the relative gene expression is fairly similar ranging from an average around 1E-05 to 4E-05. In regards to the MT_2N4 sample, the qPCR process may have failed to amplify the cDNA, hence an unreliable MT/EF1 value that was not used to analyze between diploids and triploids.

__RNA Quantification Results__ The results of the RNA quantification are listed in the following tables: The RNA concentration measured in ng/μL, was recorded along with the A260/280 and A260/230 absorbance values. Table 1 lists values for triploid wet (3nW), dry (3nD) and diploid wet (2nW), dry (2nD) C. Gigas gill tissue oysters (12 samples total). The wet were kept in the carbonated low pH seawater for the entire experiment and the dry tissues were removed 24 hours prior to sampling tissue. Table 2 lists values for 4 diploids (listed 1 - 4) and 5 triploid (listed 1 - 5) C. Gigas gill tissue oysters submerged in control pH seawater for the entire experiment.
 * Table 1 control pH**
 * High pH || RNA Concentration (ng/μL) || A260/280 || A260/230 ||
 * 3nD || 239.8 || 1.82 || 1.26 ||
 * 3nD || 723.3 || 1.91 || 1.43 ||
 * 3nD || 353 || 1.84 || 2.26 ||
 * 3nW || 1287.7 || 1.96 || 2.28 ||
 * 3nW || 608.3 || 1.91 || 2.33 ||
 * 3nW || 594.1 || 1.92 || 1.58 ||
 * 2nD || 1167.8 || 1.94 || 1.49 ||
 * 2nD || 1009.1 || 1.98 || 2.14 ||
 * 2nD || 906.2 || 1.9 || 2.15 ||
 * 2nW || 597 || 1.86 || 2.15 ||
 * 2nW || 373.6 || 1.85 || 2.05 ||
 * 2nW || 364.6 || 1.84 || 2.25 ||
 * Average || 685.375 || 1.894167 || 1.9475 ||
 * Table 2 low pH**
 * Low pH || RNA Concentration (ng/μL) || A260/280 || A260/230 ||
 * 2n(1) || 2435.4 || 1.9 || 1.2 ||
 * 2n(2) || 1097.4 || 1.94 || 1.12 ||
 * 2n(3) || 2050.4 || 1.93 || 1.22 ||
 * 2n(4) || 1866.3 || 1.97 || 1.43 ||
 * 3n(1) || 691.7 || 1.95 || 1.33 ||
 * 3n(2) || 2323.4 || 1.91 || 1.45 ||
 * 3n(3) || 1520.7 || 1.93 || 1.31 ||
 * 3n(4) || 1693.1 || 1.99 || 1.7 ||
 * 3n(5) || 1292.3 || 1.98 || 1.56 ||
 * Average || 1663.411111 || 1.944444 || 1.368889 ||

__Conclusion__ The characteristic curves generated in the Nanodrop indicated relatively good measurements. The amount of RNA concentrated recorded varied, but with relatively more RNA concentration in the low pH gill tissues versus the control pH tissues. This difference could be from the experimental conditions the control pH oysters were subjected to (dry versus wet). In regards to the A260/280 values, a clean sample should be between 1.8 - 2.0, which is characteristic of all the samples, meaning a relatively pure RNA sample of protein. The 260/230 ratio should ideally be between 1.5 - 2.0, however the majority of the samples are under or over the given range. This indicates a carryover of phenol, ethanol, or high salt. Most likely ethanol from the final phases of RNA extraction.

__Reflection__ At this point, after quantifying and knowing that qPCR works to analyze gene expression for C.Gigas gill tissue with metallothionein gene primers and EF1 gene primers, the next step is to do qPCR for the control pH. This would generate MT/EF1 values that can be compared to the low pH MT/EF1 values to note any differences in gene expressions at different pHs and on a broader scale, to ocean acidification. The 2N4 might have failed to amplify either through a lack of polymerase present, missing primers, or bad cDNA.

Date November 14, 2011

__Summary__ Using a cytosine methylation dot blot, C. gigas tissue was analyzed to see the amount of epigenetic DNA methylation. Also, quantitative PCR was run to analyze the relative gene expression of metallothioneins in C. gigas.

__Materials__
 * C. Gigas cDNA
 * 1.5 mL screw cap tubes
 * 1.5 mL microcentrifuge tubes (RNase free)
 * Lab coat and gloves
 * 6X SSC
 * Hot plate
 * Microcentrifuge
 * Beaker + microcentrifuge floaties
 * Pipettes + tips
 * Ultra filtered water
 * Blocker Diluent Part A + Part B
 * Rotary shaker
 * 5-MeC antibody
 * TBS-T
 * Secondary antibody solution
 * Chromogenic substrate
 * Nylon membrane
 * Pipette tip box
 * Filter paper
 * Scissors
 * PCR plate (white strips + clear caps)
 * Nuclease free water
 * Thermal cycler
 * 2X Immomix Master mix
 * SYTO-13 Dye

__Method__ DNA DILUTION
 * 1) Obtain C.Gigas cDNA sample tissue from previous reverse transcription - Initial concentration was 271.5 nanogram/microliter with 1.80 A260/280 ratio
 * 2) Make initial dilution of cDNA to 50 nanogram/microliter in 1.5 mL snapcap- Use equation C1V1 = C2V2. Using calculations, add 7.37 microliter DNA + 32.63 microliter H2O.
 * 3) Label 5 screw caps with initials, "DNA", and target concentrations
 * 4) Add dilution preparations for each screw cap with different target concentrations - 0.8, 0.4, 0.2, 0.1, 0.05 nanogram/microliter
 * 5) Add 60 microliter 20X SSC and 124, 132, 136, 138, 139 microliter of water to each target concentration respectively as listed
 * 6) Add 16, 8, 4, 2, and 1 microliter of initial cDNA dilution to each screw cap respective of previous target concentrations listed

DOT BLOTTING
 * 1) Cut nylon membrane to fit dot blot wells - there are 72 of them
 * 2) Soak membrane in 6X SSC for 10 min in pipette tip box
 * 3) Cut filter paper to fit nylon membrane and wet in 6X SSC
 * 4) Assemble vacuum manifold with nylon membrane on top of filter paper
 * 5) Denature DNA screw cap tubes in beaker with floaties for 10 minutes then transfer to ice.
 * 6) Switch on vacuum. Apply 500 microliter of 6X SSC to each well and allow SSC to filter through - vacuum rate should filter all SSC within a couple of minutes
 * 7) Spin down cDNA at 13200 rpm for 5 minutes
 * 8) Apply screw cap target concentrations to each well individually - column 7 A - E
 * 9) Allow samples to filter through
 * 10) Soak filter paper previously cut in denaturation buffer as samples pass through to nylon membrane
 * 11) After samples filter through, soak membrane on denaturation buffer filter for 10 minutes, then neutralization-soaked filter for 5 minutes
 * 12) Wrap dryed blot in plastic wrap and place DNA-side down on UV transluminator for 2 minutes at 120kJ.

WesternBreeze CHROMOGENIC IMMUNODETECTION
 * 1) Prepare 20mL blocking solution with 14mL ultrapure H2O, 4mL diluent A, and 2mL diluent B.
 * 2) Place membrane in 10mL blocking solution in covered plastic dish - then incubate for 30min on rotary shaker at 1 rev/sec
 * 3) Decant blocking solution
 * 4) Rinse membrane with 20mL water for 5 minutes then decant
 * 5) Repeat step 4
 * 6) Prepare 10mL primary antibody solution to 1:5000 dilution - 10mL blocking solution + 2 microliter 5-MeC antibody
 * 7) Incubate membrane with 10mL primary antibody solution for 1 hour.
 * 8) Decant primary antibody and wash membrane for 5 minutes with 20mL 1X TBS-T. Decant.
 * 9) Repeat step 8 three times
 * 10) Incubate membrane in 10mL secondary antibody solution for 30 minutes and decant
 * 11) Wash membrane for 5 minutes with 20mL TBS-T.
 * 12) Repeat step 11 three times
 * 13) Rinse membrane with 20mL water for 2 minutes - decant
 * 14) Repeat step 13 two times
 * 15) Incubate membrane in 5mL chromogenic substrate until color develops - takes ~1-60 minutes
 * 16) Rinse membrane with 20 mL water for 2 minutes and decant
 * 17) Repeat step 16 two times
 * 18) Dry membrane with clean piece of filter paper

PRIMER RECONSTITUTION PROTOCOL
 * 1) Spin down new primers for 1 minute at 13200 rpm
 * 2) Add 209 microliter TE buffer to metallothionein forward primer
 * 3) Add 255 microliter TE buffer to metallothionein reverse primer

PRIMER WORKING SOLUTION PROTOCOL
 * 1) 10 microliter of metallothionein forward + 90 microliter H2O in one 1.5mL snap cap tube. Label M-F LT 11/14/11
 * 2) 10 microliter of metallothionein reverse + 90 microliter H2O in another 1.5mL snap cap tube. Label - M-R LT 11/14/11

QUANTITATIVE PCR PROTOCOL
 * 1) Prepare master mix for 4 reactions + 1 more - 62.5 microliter 2X immomix + 5 microliter SYTO-13 Dye + 6.25 microliter upstream and downstream primer + 35 microliter H2O
 * 2) Add mastermix (23 microliter) to each white PCR plate
 * 3) Add 2 microliter thawed cDNA C.Gigas into two wells
 * 4) Add 2 microliter ultrapure water into the other two wells as controls
 * 5) Cap white strips with clear caps and label strips - LT
 * 6) Spin white strips down briefly to pool mixture
 * 7) Place strips on ice
 * 8) Load plate into thermal cycler for PCR.

__Results__ In regards to the dot blot, the results show the dye was not very effective. The negative control, the fly DNA, which should have little to no DNA methylation, had little binding which was the expected result. The human DNA however, which should have lots of DNA methylation and a high amount of antibody binding, had little binding as well. As a result, the dot blot, although had characteristic little binding to the negative control, also had little binding to the positive control and thus this may be attributed to poor antibody binding. In regards to the qPCR, there was no amplification for the tested C.Gigas cDNA metallothionein DNA region with the primers used. However, from other classmates, there was a nice single melt curve with good binding, indicating an effective means of quantifying gene expression.

__Conclusions__ A reason why the cDNA qPCR did not amplify is because of the type of metallothionein primer used. The designed primers were for a metallothionein promoter region, which was most likely not expressed in the RNA used for reverse transcription. There are a different set of primers available that have been designed off the metallothionein DNA region which will be used for future qPCR procedures. This different set of primers will more than likely exhibit results if metallothionein is expressed in the C. Gigas RNA extracted. As for the dot blot, a different antibody used might bind better to the given methylated cytosines, which may indicate better results. However, the results used in this specific experiment are not reliable enough to draw a firm conclusion.

__Reflection__ Now that the technique of dot blotting and qPCR have been shown, these techniques will be applied to experiments for the ocean acidification group. Some members will be working on dot blotting to analyze the level of DNA methylation in stressed C. Gigas in regards to ocean acidification. This might portray how epigenetics influences, or is influenced by, stressful conditions for crassostrea gigas. In terms of metallothioneins, qPCR will be used to detect the level of gene expression in C. gigas in response to high and low carbon dioxide in seawater. This will exhibit any correlation between stress responses to different stressors.

Date: November 8, 2011 + November 10, 2011

__Summary__ Using previous techniques learned in lab (RNA and protein extraction, etc), the oyster gill tissue samples from our ocean acidification experiment were prepared for RNA extraction. The extracted RNA was then prepared to undergo cDNA reverse transcription. Specifically, I extracted RNA and did reverse transcription for 9 gill samples from C. Gigas in carbonated seawater (high pH) and 9 gill samples in control seawater (low pH).

__Materials__
 * 9x C.Gigas gill seawater control samples
 * 9x C. Gigas gill carbonated seawater samples
 * 1.5mL Snap cap tubes
 * Gloves and goggles
 * Refrigerated centrifuge
 * Materials for RNA Isolation (See RNA isolation protocol October 4, 2011)
 * Materials for RNA Extraction (See RNA extraction protocol October 11, 2011)
 * Materials for reverse transcription (See Reverse transcription protocol October 18, 2011)

__Method__
 * 1) Obtain 18x samples (9x seawater control gill tissues + 9x carbonated seawater gill tissues).
 * 2) Label 18x snap cap tubes with initials, experiment group, date, and tissue type - LT low (or high) pH C. Gigas
 * 3) From each sample, obtain ~0.025 - 0.05g gill tissue and put into each tube.
 * 4) Follow RNA isolation protocol to completely extract RNA - This step was difficult since I had to work fast to homogenize tissue and store on ice to prevent RNA degradation.
 * 5) After isolating RNA, follow RNA extraction protocol - pellets obtained were ranged from very small (generally high pH samples) to relatively large (low pH).
 * 6) After RNA isolation, store on ice and follow reverse transcription protocol - master mix RT was prepared for 9 low pH samples + 1 for pipetting error. The other 9 high pH samples had their own master mix +1 sample for pipetting error.

__Results__ No results were collected for this lab. The cDNA reverse transcription process was the last thing to run, and the cDNA will then undergo qPCR in the next lab.

__Conclusions__ Due to a high volume of samples, the carbonated seawater 9x samples underwent RNA isolation, extraction, and RT first (Nov. 8 2011) before the other 9x seawater samples were extracted (Nov. 10, 2011). This was to prevent the samples from sitting at room temperature. This was to keep the RNA from degrading at room temperature due to the instability of RNA. The next process, if the cDNA is completed, is to take the samples for qPCR to determine gene expression. A metallothionein gene primer will be used on the cDNA to undergo qPCR and to see whether there is varying gene expression in either group.

__Reflection__ The purpose and techniques used in this lab are to determine whether ocean acidification elicits a general stress response "package" in crassostrea gigas. Although metallothioneins are generally associated with a xenobiotic response, it would be interesting to see if oysters upregulate different stressor genes in response to any general stress such as increased acidification. Although this experiment will not pinpoint a specific reason as to why it might upregulate different stressor genes, it is a preliminary test to better understand the nature of the stressed oyster. If results are as expected and a notable difference is apparent between different experimental groups, further tests for other stress genes can be done.

Date: November 1, 2011

__Summary__ Using the previous PCR products, Gel Electophoresis was conducted to assess the purity and accuracy of the PCR product. Also, protein was extracted from research project sample tissues to undergo protein analysis in SDS-Page and western blot techniques.

ELECTROPHORESIS PROTOCOL

__Materials__
 * 1.5% Agarose gel with ethidium bromide
 * 1x TAE buffer
 * 100bp ladder
 * PCR product from last week's lab
 * Voltmeter and gel electrophoresis apparatus
 * UV transilluminator

__Method__
 * 1) Place gel in box and add 1x TAE buffer to cover wells
 * 2) Remove comb from wells
 * 3) Load 7 microliter 100bp ladder in far left lane
 * 4) Load 25 microliter PCR products into adjacent lanes. There should be 4: 2 samples and 2 controls
 * 5) Run gel at 100V for 1 hour
 * 6) Analyze purity and results on UV transilluminator

PROTEIN EXTRACTION PROTOCOL

__Materials__
 * 0.023g C. Gigas Mantle Tissue
 * 1.5mL Snap cap tube
 * CellLytic MT Solution
 * Disposable Pestle
 * Gloves and Goggles
 * Centrifuge
 * 1000 microliter pipette and pipette tips

__Method__
 * 1) Obtain 0.023g C. Gigas Mantle Tissue and put into 1.5mL snap cap tube.
 * 2) Label snap cap tube with initials, tissue, date - 0.023g C. Gigas Mantle LT 11/1/11
 * 3) Add 500 microliters of CellLytic MT to the snap cap tube with tissue sample.
 * 4) Homogenize tissue sample with disposable pestle
 * 5) Close the snap cap tube and invert several times to mix
 * 6) Centrifuge the tissue sample at 13300 rpm for 10 minutes
 * 7) Label a new 1.5mL snap cap tube with the word "Protein", tissue, initial, and date - Protein C. Gigas Mantle LT 11/1/11
 * 8) Transfer supernatant from centrifuged tube to new "Protein" snap cap tube
 * 9) Store "Protein" tube with supernatant on ice

SDS-PAGE PROTOCOL AND WESTERN BLOT PROTOCOL

__Materials__
 * Hot plate
 * Protein extract in 1.5mL snap cap tube
 * 1.5mL Screw cap
 * 2x Reducing sample buffer
 * Western blot xcell solution box with electrodes and voltmeter
 * Nitrocellulose membrane
 * Filter paper
 * Western blot agarose gel
 * Nanopure water
 * Blocking solution
 * Rotary shaker
 * Primary antibody solution
 * Antibody wash
 * Secondary antibody solution
 * Tris-Glycine 1x transfer buffer

__SDS-PAGE Method__
 * 1) Boil water on hot plate
 * 2) Invert protein extract tube several times and add 15 microliter to 1.5mL screw cap. Return protein extract to ice and label screw cap - Protein LT 11/1/11
 * 3) Add 15 microliter 2x reducing sample buffer.
 * 4) Mix sample by flicking and centrifuge at 9000rpm for 10 seconds.
 * 5) Boil sample for 5 minutes then immediately centrifuge for 1 minute at 9000rpm
 * 6) Load sample into western blot wells that have transfer buffer in them already
 * 7) Set up the gel box with transfer buffer and attach to a power supply.
 * 8) Run the gel at 150V for 45 minutes
 * 9) Turn off power supply and disconnect gel box from power supply.
 * 10) Remove gel box lid, disconnect tension wedge, and remove gel from gel box.
 * 11) Open casette to expose gel.
 * 12) Trim wells on gel top.
 * 13) Notch a designated corner (upper right) to remember gel orientation.
 * 14) Take gel and put through western blot protocol.

__WESTERN BLOT PROTOCOL__
 * 1) Soak filter paper, nitrocellulose membrane and gel in 1x Tris-Glycine transfer for 15 minutes.
 * 2) Assemble blotting sandwich in this order from top to bottom: + Anode, filter paper, membrane, gel, filter paper, - cathode.
 * 3) Run blotting sandwich at 20V for 30 minutes.
 * 4) Remove gel from sandwich and put into Cromassie brilliant blue reagent.
 * 5) Wash membrane 2 times for 5 minutes each with 20mL nanopure water. Put on rotary shaker for each of the 5 minutes.
 * 6) Create the primary antibody solution by diluting with blocking solution 1:3000. This is 10 mL blocking solution and 4 micrograms primary antibody.
 * 7) Put membrane in plastic box and add 10 mL blocking solution.
 * 8) Decant Cromassie brilliant blue reagent on gel and add ~10mL nanopure water.
 * 9) Cover and incubate membrane in plastic box overnight on rotary shaker at 1 revolution/second.
 * 10) Cover and incubate the nitrocellulose gel in another plastic box on the rotary shaker at 1 revolution/second too.

__Results__ The Gel Electrophoresis produced 3 solid, visible bands in two sample and one control well. The fourth control band produced a fainter band. All were around ~200bp according to the ladder. The Western Blot produced visible bands in regards to the sea urchin HSP70 protein extracted, however there were no visible bands for oyster HSP70.

__Conclusion__ Based upon gel electrophoresis product analysis in the UV transilluminator, the PCR products were all around ~200bp which is consistent with the product size of the primer used: Gigasin-2 F and R for HSP70. As for the controls, one was clearly contaminated with a visible, thick band around the same area as the sample bands. The other control was much fainter, but could also be through contamination as well as primer dimerization, since the band ran a little farther down the gel, indicative of a smaller DNA strand. In regards to the Western Blot, a few things could be interpreted of the results. One is that the antibodies used to detect protein were not specific for oyster HSP70 but rather sea urchin HSP70. Hence, no visible band would be produced for the oyster Western Blot. Another interpretation is that there was no HSP70 protein expressed in the oyster for the antibody to bind to. Further tests must be taken, such as a different, oyster specific antibody or another detection technique.

__Reflection__ This lab has demonstrated essential and fundamental laboratory techniques for DNA, RNA, and protein analysis. Gel electrophoresis demonstrated a mechanism to assess purity and product size of a PCR reaction to determine whether or not the targeted gene was indeed the one being amplified in PCR. Also, SDS-Page and Western blot analysis allows for specific protein analysis. These two techniques can add to specificity when analyzing proteins, since they work to pinpoint and target a specific protein with the use of antibodies and the antibodies of antibodies.

Date: October 25, 2011

__Summary__ Using the cDNA prepared from the previous lab and primers, the cDNA was prepared for PCR using a Thermocycler. PCR is used to replicate the cDNA strand for the gene of interest for further analysis, such as sequencing the gene.

PCR PROTOCOL

__Materials__
 * Micropipettes with pipette tips (20, 200, 1000 microliters)
 * 0.5mL PCR tubes
 * 1.5mL microcentrifuge tubes (RNAse free)
 * cDNA sample (C. Gigas)
 * dNTPs
 * 5x GoTaq Green buffer
 * Primer (S. White 43671961 Gigasin-2 Reverse + Forward)
 * Nuclease Free Water
 * Thermocycler
 * Microcentrifuge
 * Gloves and goggles
 * Waterbath
 * TE Buffer

__Method__
 * 1) Prepare primer by centrifuging for 3 minutes at 7500 rpm.
 * 2) Add 269 microliters of TE buffer.
 * 3) Warm for ~1 minute in 45C waterbath then vortex briefly (~5 seconds)
 * 4) Centrifuge for 3 minutes at 7500 rpm and vortex briefly again.
 * 5) Label a 1.5mL microcentrifuge tube with "MM" and initials - MM LT/AT
 * 6) Add 125 microliter 5x GoTaq Green buffer, 5 microliter forward and reverse primer each, and 105 microliter nuclease free water into MM tube. This is your master mix.
 * 7) Label 4, 0.5mL PCR tubes with sample and initials - C. Gigas LT/AT
 * 8) Pipette 48 microliters of master mix into each 0.5mL PCR tube.
 * 9) Add 2 microliters of cDNA sample into 2 of the 0.5mL PCR tubes.
 * 10) Add 2 microliters of water into the other 2 0.5mL PCR tubes. Label these as "C" for control - C
 * 11) Briefly centrifuge tubes to pool liquid at the bottom.
 * 12) Secure the caps on the PCR tubes and place into thermocycler.
 * 13) Store samples at 20C after thermocycler.

__Conclusion__ Nothing was measured in this lab, but the following cDNA PCR will allow for the gene of interest, amplified from the specific primers used, to be analyzed in any format - sequencing, electrophoresis for size, etc.

__Reflection__ The purpose of this lab is to teach the fundamentals of PCR. This lab demonstrated the techniques of PCR preparation, the methodology, and the rationale behind DNA amplification. PCR is a useful and insightful scientific technique that allows for further investigations into animal physiology. It aids in understanding animal behavior, response, and function at a molecular and genetic basis by referring to the roots of the response: the DNA. Further questions I have about this lab is the type of Taq polymerase used, and whether there are different types that should be used for different organisms.

Date: October 18, 2011

__Summary__ The RNA extracted from the previous lab will be converted to cDNA using reverse transcription. Also, we further discussed designing an experiment with ocean acidification and oysters, as well as discussing primer design.

REVERSE TRANSCRIPTION PROTOCOL

__Materials__
 * Micropipettes
 * 0.5 mL PCR tubes
 * RNA sample
 * M-MLV Reverse Transcriptase
 * M-MLV 5X Reaction buffer
 * Oligo dT
 * dNTPs
 * DI Water
 * Thermal cycler
 * Microcentrifuge tubes
 * Ice bucket
 * Gloves

__Method__ > PRIMER DESIGN
 * 1) Thaw and mix RNA sample after removing from stored ice.
 * 2) Label 0.5mL PCR tube with word "cDNA" and initials - cDNA LT/IS
 * 3) In 0.5mL PCR tube add: 0.5 microliter RNA, 1 microliter oligo dT, and 4 microliter DI water.
 * 4) Incubate PCR tube for 5 minutes at 70 degrees Celsius in thermocycler.
 * 5) Immediately transfer to ice for 2 minutes.
 * 6) Vortex briefly to mix and briefly desk top centrifuge for ~5 seconds
 * 7) After centrifugation, add 5 microliter M-MLV 5X Reaction buffer, 5 microliter dNTP, 1 microliter M-MLV RT, 4 microliter DI water.
 * 8) Incubate for 60 minutes at 42 degrees Celsius and then heat inactivate for 3 minutes at 70 degrees Celsius.
 * 9) Spin down sample in desk top centrifuge.
 * 10) Store on ice at -20 degrees Celsius.
 * 1) Conduct research on C.Gigas and determine genes in stress response - HSP70
 * 2) Using the NCBI Databank, find the gene of interest and design a primer.
 * 3) Post on Google Docs

__Conclusion__ Nothing was measured from this lab, but from the reverse transcription process, primers will be attached to the cDNA to undergo PCR for cDNA analysis.

__Reflection__ The purpose of this lab is to understand that RNA for specific proteins can be extracted, and using a backwards process, turn the RNA back into its original DNA sequence through complimentary DNA reverse transcription. This allows for comparison of the cDNA to the location of the gene on the chromosome, allowing for further understanding of how and when an RNA (or protein) is expressed. These methods are very effective in finding more infomation about new proteins and RNA discovered.

Date: October 11, 2011

__Summary__ Using the C.Gigas Gill tissue prepared in the previous lab, the RNA content of the tissue was extracted and using a Nandrop spectrophotometer, the RNA concentration, the A260/280 ratio, and the A260/230 ratio was obtained.

RNA EXTRACTION PROTOCOL

__Materials__ __Method__
 * Micropipettes (10, 20, 200, 1000 microliter tips and pipettes)
 * 1.5mL microcentrifuge tubes
 * Gloves, goggles, labcoat
 * Timer
 * Ice buckets
 * Phenol
 * Chloroform
 * Vortex
 * Nanodrop spectrophotometer
 * 75% EtOH
 * Isopropanol
 * 0.1% DEPC Treated water
 * 1) Incubate C.Gigas Gill tissue sample from Lab 1 at room temperature for 5 minutes. Tube containing the tissue has the gill tissue at the bottom in a pink solution.
 * 2) Add 200 microliters of chloroform to the tissue in the fume hood.
 * 3) Vortex tissue for 30 seconds. Sample becomes a white, cloudy mixture.
 * 4) Incubate tissue for 5 minutes at room temperature.
 * 5) Centrifuge tissue in refrigerated microfuge for 15 minutes at max speed.
 * 6) Transfer the upper, aqueous phase of the centrifuged sample to another microcentrifuge tube, but not the interphase or organic phase. Interphase is a white, thin layer between the upper aqueous and lower organic phases.
 * 7) Add 500 microliter isopropanol to the new microcentrifuge tube with the aqueous phase (contains the RNA) and invert several times to mix until solution appears uniform. Should invert at least 5-8 times.
 * 8) Incubate tube for 10 minutes at room temperature.
 * 9) Spin in refrigerated microfuge at max speed for 8 minutes. A cloudy white pellet should form at the bottom of the tube. Ours was very small, almost a speck.
 * 10) Remove the supernatant from the tube and add 1mL of 75% EtOH to pellet. Vortex to dislodge the pellet if needed. In our case, we did not need to vortex at this step.
 * 11) Centrifuge for 5 minutes at 7500g.
 * 12) Remove supernatant. If needed, briefly spin tube to pool residual EtOH to remove. We used a 20 microliter pipette to remove residual EtOH and let the pellet sit at room temperature to evaporate the rest of the EtOH instead: ~3 minutes.
 * 13) Add 100 microliter 0.1% DEPC Water to pellet and pipette up and down to dissolve pellet in solution.
 * 14) Flick tube a couple of times with finger to mix and place on ice.
 * 15) Quantitate RNA with Nanodrop spectrophotometer.

RNA QUANTIFICATION


 * 1) Prepare Nanodrop by pipetting 2 microliter 0.1% DEPC Water into pedestal and lower the arm.
 * 2) Click "Blank" to zero instrument.
 * 3) Pipette 2 microliter RNA sample onto Nanodrop pedestal and lower arm.
 * 4) Click "Measure". Record RNA concentration, A260/280 ratio, and A260/230 ratio. RNA Concentration: 102 nanogram/microliter. A260/280 Ratio: 1.70. A260/230 Ratio: 1.87.
 * 5) Raise Nanodrop arm and wipe with KimWipe.
 * 6) Label stock RNA sample with the word "RNA", source organism/tissue, initials, date, and concentration - RNA C.Gigas Gill Tissue, LT/IS, 10/11/11, 0.102 microgram/microliter.
 * 7) Give sample to TA to store at -80C.

__Conclusions__ Based upon our results, the RNA concentration of 102 nanogram/microliter is fairly good. Although when compared to the RNA concentrations of other classmates, this number is quite low, with some groups obtaining measurements in the 1000 range. Nevertheless, this is a good amount of RNA obtained. Analyzing the A260/280 and A260/230 ratios, a clean RNA isolation should have ratios of 1.8-2.0 and 1.5-2.0 respectively. Our A260/230 ratio is within the 1.5-2.0 range, at 1.87, however our A260/280 ratio of 1.70 is below the range of 1.8-2.0 for a good sample. The A260/230 ratio range indicates a clean RNA sample with respect to phenol, ethanol, or high salt, all of which absorb around 230nm, so our RNA extraction was relatively clean of these compounds. However, the A260/280 ratio indicates proteins, which absorb around 280nm. The next step at this point of obtaining the RNA is to create its complementary DNA and potentially sequence it to determine the specific RNA extracted.

__Reflections__ The purpose of the lab was to familiarize the Nanodrop spectrophotometer technique in determining RNA concentration in tissue samples. The lab taught us to prepare tissues using incubation, centrifugation, and different compounds to isolate the RNA and utilize its absorbance properties to determine the RNA concentration. This lab is potentially useful since it allows for RNA extraction, which enables further research upon physiological responses of organisms; either through sequencing the RNA, its cDNA, or determining what type of protein the RNA is translated into.

Date: October 4th, 2011

__Summary__ The first part of this lab is the preparation to extract RNA from a given tissue. TriReagent will be used to help isolate RNA from the given tissue. The second part is protein extraction from a given tissue sample. CellLytic MT solution will be used to expose proteins, and Bradford reagent will be used to determine the absorbance of the tissue sample which corresponds to a particular protein concentration calculated from a standard curve of protein concentration vs. absorbance.

RNA ISOLATION PROTOCOL

__Materials__
 * 1.5mL Snap cap tube
 * Lab marker
 * TriReagent
 * 0.013g C.Gigas gill tissue
 * Ice
 * Disposable Pestle
 * Vortex
 * Gloves
 * 1000 microliter pipette

__Method__
 * 1) Once tissue is obtained, label 1.5mL snap cap tube with date, initials, and tissue information - 10/4/11 LT 0.013g C.Gigas gill. The tissue is orange and rubbery in texture.
 * 2) Put tissue into snap cap tube and then store on ice.
 * 3) Add 500 microliters of TriReagent into snap cap tube and mash tissue with disposable pestle until homogenized. The tissue was relatively small and hard to mash with the head of the pestle. Thus, after mashing with the pestle, the sample was vortexed for at least 7 seconds.
 * 4) Add another 500 microliters of TriReagent and tightly close snap cap tube. Vortex for another 15 seconds.
 * 5) Tissue is labeled and ready to store at -80C for lab 2.

PROTEIN EXTRACTION AND ANALYSIS PART I

__Materials__ __Method__
 * 0.007g C. Gigas mantle tissue
 * 1.5mL Snap cap tube
 * CellLytic MT solution
 * Disposable Pestle
 * Centrifuge
 * 2mL Screw cap tube
 * DI Water
 * 20 microliter pipette
 * 1000 microliter pipette
 * Bradford reagent
 * 1000 microliter Cuvette
 * Spectrophotometer
 * Gloves
 * 1) Obtain tissue and label 1.5mL snap cap tube with initials, date. Record weight and information of tissue in lab notebook, then put tissue into snap cap tube - LT 10/4/11 – 0.007g C.Gigas Mantle.
 * 2) Add 500 microliters of CellLytic MT Solution to snap cap tube and mash with disposable pestle to homogenize. This tissue is relatively easy to mash up. The orange tissue broke down into little pieces and the solution in the snap cap tube became cloudy.
 * 3) Close tube and invert several times to mix.
 * 4) Centrifuge in a refrigerated microfuge for 10 minutes at max speed.
 * 5) Label a new, fresh tube with the word “Protein”, tissue information, initials, date - Protein 0.007g C. Gigas Mantle LT 10/4/11.
 * 6) Take centrifuged snap cap tube and transfer supernatant to new labeled tube and store on ice.
 * 7) Label a fresh 2mL screw cap tube with “Protein”, BA, initials, date - Protein BA LT 10/4/11.
 * 8) Dilute protein sample in half by pipetting 15 microliter of protein sample into 2mL screw cap tube with 15 microliter DI water then mix well.
 * 9) Add 30 microliter DI water into second 2mL screw cap tube. Label tube as “blank”.
 * 10) Add 1.5mL Bradford reagent to both screw cap tubes.
 * 11) Invert both tubes several times and incubate at room temperature for ~10minutes.
 * 12) Mix blank tube via pipetting and transfer to cuvette. Solution is a dark blue, clear solution
 * 13) Zero spectrophotometer with blank.
 * 14) Mix sample tube via pipetting and transfer to cuvette. Solution is a darker blue, clear solution
 * 15) Measure absorbance at 595 nm and record value. Absorbance: 0.111
 * 16) Repeat steps 14 – 15 for a second measurement. Absorbance: 0.114
 * 17) Average both absorbance values and determine protein concentration from given standard curve with equation y=1013.9x. In this case, x=absorbance, and since there was a dilution in step 8, must multiply the average absorbance by 2. Thus, true absorbance is equal to 0.228. Substitute x in the equation for 0.228, and the calculated protein concentration for the given C.Gigas mantle tissue is 231.17 micrograms/mL.

__Conclusion__ After further analysis, the obtained results from our experiment seem reasonable. A proper absorbance was measured from the given samples and a reasonable protein concentration was measured. From the given results, the next that can be taken is a comparison with fellow classmates in regards to their obtained protein concentrations. Comparisons can be made with other groups that measured mantle tissue, as well as with other types of tissues like C.Gigas gill tissue.

__Reflection__ The purpose of this lab was to familiarize us in techniques to determine protein concentration using a spectrophotometer and Bradford reagent. On a broader scale, this experiment can apply to measuring a concrete variable in regards to different experimental designs. Protein concentration is a valid measurement that can be made in experiments that test an organism’s response to physiological stress. For instance, the effect of temperature on an organism can be measured through the various levels of protein concentrations within its body. What still remains a bit unclear in the experiment is the impact of homogenizing the tissue sample. Would there be more absorbance, and thus more concentration, if the tissue was completely homogenized in the solution?