Tuesday, December 7th, 2010:
This week the group finished working on their personal data collection, paper, and paper review of the other group. Because I ran my qPCR late last week, most of my effort was put towards working on the group paper last weekend and reviewing the salmon group's paper. In addition, after another group member ran the housekeeping gene as a reference I was able to calculate my Fold/Min Normalized Gene Expression Value and the data is below:


data.JPG
Excel Data to calculate fold/min normalized gene expression value


hsp70_graph.JPG
Average gene expression in fold per min of HSP70 in control, V. tubiashii, copper, and V. tubiashii + copper treatments.


NOTE: some values in column AC and AD were a little off, they have been corrected on the Google Sheet - sr320 sr320 Dec 12, 2010


After viewing the comments on our paper, the remainder of my time will be to finish up and revise my sections of the paper as well as work on my ppt slides for our presentations on Wednesday.

It's been a fun quarter and I've learned a lot! Thanks for a great quarter and all the help with our projects!

Tuesday, November 30th, 2010:
RNA was extracted and quantified from half of the C. gigas gill tissue/per animal (the other half was used for Paul's protein extraction) using the following protocol:


RNA Extraction

      1. Turn on heating block and heat to 55°C.
      2. Incubate previously isolated RNA tissue sample tube at room temperature for 5 minutes.
      3. In the fume hood, add 200 uL of chloroform to sample making sure to close the tube before removing from fume hood.
      4. Vortex sample for 30 seconds so the sample solution becomes a milky emulsion.
      5. Incubate sample at room temperature for 5 minutes.
      6. Spin tube in refrigerated microfuge for 15 minuets at max speed.
      7. Gently remove tube from microfuge making sure to not to disturb the tube.
      8. Transfer most of the aqueous phase (top, clear portion) to a fresh microfuge tube labeled "Sam 10/12 RNA Ext C. gigas"
      9. Quickly close the tube containing the organic and interphase and properly dispose of the liquid inside the tube as well as the tube itself in proper chloroform disposal.
      10. Add 500uL isopropanol to the tube containing RNA and cap the tube.
      11. Invert the tube several times until the solution appears uniform (it should no longer appear lumpy).
      12. Incubate at room temperature for 10 minutes.
      13. Spin in refrigerated microfuge at max speed for 8 minutes making sure to place the tube in the microfuge so the tube is hinge pointing up, away from the center of the microfuge (this allows for optimal pellet formation).
      14. Remove supernatant making sure not to disturb RNA pellet.
      15. Add 1 mL of 75% EtOH to pellet.
      16. Close tube and vortex briefly to dislodge pellet from the side of the tube.
      17. Spin sample tube in refrigerated microfuge at 7500 g for 5 minutes.
      18. Carefully remove supernatant making sure again to not disturb pellet.
      19. Briefly spin tube for about 15 seconds.
      20. Pipette remaining EtOH.
      21. Leave tube open to allow pellet to dry at room temperature for no more than 5 minutes.
      22. Add 100 uL of 0.1% DEPC-H2O to sample tube and resuspend pellet by pipetting up and down.
      23. Incubated tube at 55°C for 5 minutes.
      24. Remove tube from heat, flick to mix, and place stock RNA sample on ice until quantified using Nanodrop spectrophotometer.

RNA Quantification

      1. To blank instrument, pipette 2µL of 0.1%DEPC-H20 onto the Nanodrop pedestal and lower the arm and click blank on computer program.
      2. Wipe instrument with KimWipe, pipette 2 µL of RNA sample onto the Nanodrop pedestal and lower the arm, and click measure.
      3. Record your RNA concentration, A260/280 ratio, and A260/230 ratio.
      4. Raise the instrument's arm and wipe off sample with a KimWipe.
      5. Store RNA sample at -80°C.


RNA concentrations ~ 3000 ng/µL. Using (C1)(V1)=(C2)(V2), we normalized our samples by calculating how much concentrated RNA is needed to make a 500 µL of 200 ng/µL RNA stock from our initial concentrations.Because our samples were still very high in RNA concentration, we DNased 50 µL of all samples, individually, by following the Turbo Free DNase kit protocol in hopes to get rid of genomic DNA and other RNA contaminants left in our samples. Following this, the Nanodrop spectrometer was used to find the concentration of our samples again. Below are our results:














































Sample Number
 Control
[RNA] (ng/µL)
 Cu
[RNA] (ng/µL)
 Vt
[RNA] (ng/µL)
 Cu+Vt
[RNA] (ng/µL)
1 A
 769.9
 812.7
 1479.1
 1226.0
2 A
 1355.2
 1304.7
 1297.8
 1403.1
3 A
 930.7
 1319.8
 1649.6
 1382.1
4 A
 817.0
 1764.3
 1387.0
 (no sample)
1 B
 1119.8
 940.8
 863.1
 885.5
2 B
 594.2
 1567.1
 1454.5
 1052.8
3 B
 1514.8
 1941.4
 2214.9
 1529.2
4 B
 (no sample)
 1524.1
 1025.8
(not much sample is left
but there's plenty of RNA
in what we have)
 1030.1


With our 260/280 falling between 1.88-2.02, most around 1.91.

A final normalization step was taken for a final end concentration of 2 ug using the following equations:

1. (RNA conc ng/uL)/1000= RNA conc ug/uL
2. (RNA conc ug/uL) * X = 2 ug ---> X = 2 ug/(RNA conc ug/uL) where X equals the amount of stock RNA to add to the reverse transcription for RNA

The following google doc is the data and calculations for each sample: https://spreadsheets.google.com/ccc?key=t7fhoi_PCVo_3vcUnIIPQAA&hl=en#gid=0
*Note because final RNA amounts vary for each sample the amount of nuclease free H2O will vary as well and was calculated by water=(5-RNA)+4 and these calculations are included in the above spreadsheet as well*

All samples were reverse transcribed using the following protocol:

  1. Thaw and mix RNA stock tube by inverting
  2. Add pre-calculated stock RNA from google doc, 1 μl oligo dT, and individual amount of nuclease free H20 based off of RNA amount added in a 0.5 ml PCR tubes (also in google doc)
  3. Return RNA stock sample to ice
  4. Incubate mixture for 5 min at 70°C on the thermocycler then put tube on ice till cool.
  5. Briefly centrifuge sample tube
  6. Add 5 μl of M-MLV 5X Reaction Buffer, 5 ul of dNTPs, 1 μl of M-MLV RT, 4 μl of nuclease free H2O to sample tube.
  7. Incubate mixture for 60 minutes at 42°C and then heat inactivate at 70°C for 3 minutes on the thermocycler.

After reverse transcription each sample was transferred to its own individual labeled 1.5 mL microcentrifuge tube and an additional 225 uL of nuclease free water was added for a final dilution so enough product is available for qPCR's of three different genes in duplicates

All samples were returned to the -20°C for storage.

On Thursday, I set up my qPCR to examine HSP70 gene expression in C. gigas in the presence and absence of Vt and Cu using our previously transcribed cDNA samples using the following ingredients:

For a 25μl reaction volume:
Component
 Volume
 Final Conc.
Master Mix, 2X (Immomix)
 12.5µL
 1x
Syto-13 dye (50uM)
 1µL
 2µM
HSP 70 forward primer, 10μM
 1.25μl
 2.5μM
HSP 70 reverse primer, 10μM
 1.25μl
 2.5μM
Ultra Pure Water
 7uL
 NA

2 uL cDNA was used as a template, while 2 uL pure H20 was used as a negative control in the following plate map:


 1
 2
 3
 4
 5
 6
 7
 8
A
 Control 1A
 Control 2A
 Control 3A
 Control 4A
 Control 1B
 Control 2B
 Control 3B
 NO SAMPLE
B
 Vt 1A
 Vt 2A
 Vt 3A
 Vt 4A
 Vt 1B
 Vt 2B
 Vt 3B
 Vt 4B
C
 Cu + Vt 1A
 Cu + Vt 2A
 Cu + Vt 3A
 NO SAMPLE
 Cu + Vt 1B
 Cu + Vt 2B
 Cu + Vt 3B
 Cu + Vt 4B
D
 Cu 1A
 Cu 2A
 Cu 3A
 Cu 4A
 Cu 1B
 Cu 2B
 Cu 3B
 Cu 4B
E
 Negative Control 1
 Negative Control 2
 Negative Control 3
 Negative Control 4




Results: I briefly looked at my qPCR results and there looks to be amplification in all samples except the negative control. Two samples (one from Cu and on from Cu+Vt) have a low melting point. I am waiting on another group member to run a plate with the house keeping gene as a reference to analyze the data from my run.

Lastly, the experiment set-up was bleached and broken down early in the week.


Tuesday, November 23rd, 2010:

Snow week!! Most of us were out for the week do to the weather. I believe Dave was able to work on processing some of his Vt challenge data but all other samples remained in storage until we are able to process them after the holiday. Happy Turkey Day!


Tuesday, November 16th, 2010:

Lab time was used to prep the experiment and make sure all supplies were available. Also Dave, David, and I started making up the LB for the Vt challenge.

This is week is the week animals are subjected to Cu, Vt, or Cu+Vt treatments. Oysters will be challenged for either 72 hrs in Cu or 48 hrs followed by an additional 24 hrs with Vt. The following is a visual outline of our plan (created by Dave):


Slide1.jpg
Slide1.jpg


LB was made for Vt dosing using the following protocol:
10 g NaCl
10 g Bactotryptone
5 g Yeast Extract
Mix on stir plate up to 800 ml with D/W
pH to 7.0
Add D/W to 1 L
Autoclave

130 ppm Cu was added to each Cu treatment on appropriate days (it was supposed to be 1.3 ppm because it is EPA relevant but I made a math error and the I/group didn't realize it until I had dosed the animals).

Because of slow Vt growth, Vt and Vp cultures were combined by Dave into one 500ml flasks of LB

Vt dosing and sampling were done on time.

Sampling prodcedure was as follows:

Tasks
Oyster Shucker - Ross for the 1st half followed by Jason
Gill tissue - me
Hemolymph - David
Mantle - Paul
Size measurements - Jason
Water filtering - Dave

Sampling over all went well. We did have two animals that were dead, but they had appeared to have been dead for some time and not due to our challenge. Some Cu treatment animals have a visibly green mantle. All Cu treatment animals had very green shells.

Tuesday, November 9th, 2010

This week we formed our groups for our class project. I am in the oyster group and I will be working on examining HSP 70 gene expression in C. gigas in the presence of Cu, Vt, and Cu+Vt. Ross, Dave, and I set up the experimental system in the basement Wednesday after class.

November 11-12th: We collected ~ 40 animals from Big Beef Creek and Belfair State Park for our experiment between 3 and 4 pm. Animals were kept in Steven's truck in coolers and added to the experimental tanks Friday morning.

Proposal


Introduction
Oysters are a very economically and ecologically important species in Washington state. Non-native C. gigas farmed animals are the number one shellfish resource for the state and produced over $57, 750, 000 in 2000 alone. Previous studies have supported that increased CO2 levels will most likely have detrimental effects on calcifying marine organisms, particularly at the larval stage (Talmage 2009). Increased quantities of CO2 will most likely affect metamorphosis and rearing abilities of calcifying organisms. Specifically, C. gigas has shown slower development rates in the presence of higher CO2 levels (Talmage 2009). When reared under high CO2 conditions (pH 7.4), more than 70% of C. gigas larvae developed without a shell or only had partial shell (Kurihara 2008). With large scale global climate changes threatening the future health of our oceans, an experiment studying the negative effects ocean acidification can have an important species is a very powerful tool to inform the public.


Material and Methods

Collection
C. gigas animals could be collected from numerous different sites around the Puget Sound region (n=30/site) that have demonstrated different pH levels. Hood Canal, for instance, has demonstrated recent pH levels as low as 7.6 and could be used as our most acidic field site. More ambient sites could be used as a natural control.
Laboratory C02 Exposure
Animals in two treatments/site (n=5/bucket resulting in 2 replicates/site/treatment) would be exposed to 700 ppm and 1000 ppm CO2 for a 2050 and 2100 predictions. Another control treatment would be held in replicates for each site (n=5/bucket). Water temperatures would be held consistent, but if we wanted a secondary stressor we could increase the number of treatments and include warmer water temperatures as that secondary stresor.
qPCR
Gill and mantle samples would be sampled and tested for gene expression in general stress response genes: HSP 70 and CytP450. RNA from samples would be isolated, normalized, and reverse transcribed into cDNA. cDNA would then be used as a template for a qPCR reaction.


Timeline

The first week would be dedicated to oyster collection. Week two would be the start of C02 exposure, which would last until the end of week 3 when sampling would occur. Week 4 would be for lab work and data analysis. Writing can be done at all stages.


References




  1. Talmage, S.C. and Gobler, C.J. 2009. The effects of elevated carbon dioxide concentrations on the metamorphosis size, and survival of larval clams (Mercenaria mercenaria), bay scallops (Argopecten irradians), and Eastern oysters (Crassostrea virginica). Limol. Oceanogr. 54(6): 2072-2080.
  2. Kurihara, H. 2008. Effects of Co2-driven ocean acidification on the early developmental stages of invertebrates. Marine Ecology Progress Series. 373: 275-284.


Tuesday, November 2nd, 2010:Quantitative PCR and Epigenetics (continued)


Summary




Materials and Methods



    1. Prepare 20 mL of Blocking Solution with the following:
      • a. Ultra filtered Water 14 ml
      • b. Blocker/Diluent (Part A) 4 ml
      • c. Blocker/Diluent (Part B) 2 ml
      • d. Total Volume 20 ml




2. Place membrane we prepared from last week in 10 ml of Blocking Solution in a plastic dish and cover

9. Incubate membrane in 10 ml of secondary antibody solution for 30 mins. Decant antibody solution.

10. Wash the membrane for 3 mins with 20 ml of TBS-T. Decant and repeat two more times
11. Rinse the membrane with 20 ml of water for 2 minutes and decant.
12. Incubate the membrane in 5 ml of Chromogenic Substrate until color begins to develop. (Color started to develop ~15 minutes after class)




Primer Reconstitution



    1. We needed to rehydrate our primers that arrived. In order to do so the nM measurement needs to have the decimal moved one decimal to the right and that amount of sterile water was added to each primer based on it's individual nM. This now is a 100 uM stock.
    2. We then created a 1:10 primer dilution by mixing 10ul primer and 90ul water in a new sterile tube.

qPCR



  1. Prepare master mix: Prepare enough master mix for SEVEN reactions to ensure sufficient volume recovery.

      1. For a 50μl reaction volume:

















Component
 Volume
 Final Conc.



Results



Conclusions
















Tuesday, October 26th, 2010: Epigenetics

Summary




Materials and Methods

Gel Electrophoresis


    1. Place previously made gel in gel box and cover with 1x TAE buffer
    2. Remove combs from wells
    3. Load 7uL 100 bp ladder in far left lane for visual aid

external image 20101027-ew4rmpw58mfcddr5tmrum6p737.jpg









DNA Dilution


    1. Prepare the following dilutions of your DNA (I signed up for the grey whale DNA)





Dilution
TARGET
      • concentration
 ul of H20
ul of 20X SSC
ul of 50ng/ul
      • DNA sample



RNA Quantification

      1. To blank instrument, pipette 2µL of 0.1%DEPC-H20 onto the Nanodrop pedestal and lower the arm and click blank on computer program.
      2. Wipe instrument with KimWipe, pipette 2 µL of RNA sample onto the Nanodrop pedestal and lower the arm, and click measure.
      3. Record your RNA concentration (=736.6 ng/µL), A260/280 ratio (=1.94), and A260/230 ratio (=1.57).
      4. Raise the instrument's arm and wipe off sample with a KimWipe.
      5. Store RNA sample at -80°C.



Dot Blotting

        1. Cut nylon membrane to fit the 72 wells of manifold
        2. Cover and soak nylon membrane in 6X SSC for 10 min
        3. Cut filter paper to the same size of the nylon membrane and wet using 6X SSC
        4. Assemble manifold with the membrane on top of filter paper
        5. Boil DNA in water for 10 minutes to denature and then transfer to ice to cool
        6. Turn on vacuum.
        7. Pipette 500 ul 6X SSC into each well and filter through.
        8. Centrifuge DNA for 5 minutes
        9. Map out samples
        10. Transfer all DNA to individual wells making sure not to touch membrane
        11. Filter DNA completely
        12. Soak cut filter in denaturation buffer
        13. Once filtration is complete, dismantle manifold and transfer membrane to filter paper soaked in denaturation buffer
        14. Let sit for 5 minutes
        15. Dry membrane on dry filter paper
        16. Wrap dried membrane in plastic wrap
        17. With DNA-side-down, view membrane on UV transluminator for 2 minutes at 120kJ to immobilize DNA







Results

PCR Gel Map
PCR Gel Map



Conclusions


















Tuesday, October 19th, 2010: Reverse transcription and end-point PCR



Step

Temperature

Time

Cycles

        1. Stored at -20°C.



        1. Mix 2g of agarose with 150mL 1x TAE in a 1L flask
        2. Microwave solution for ~ 3 minutes swirling occasionally
        3. Cool solutionand add 12 uL ethidium bromide (EtBr) and mix by swirling
        4. Pour into gel tray, add gel combs, and allow to set and cool
        5. After gel is set, wrap in plastic and foil wrap and place in fridge for next week's lab.








Tuesday, October 12th, 2010: RNA Extraction and Protein Analysis, Part 2








Protein Gel Key:

external image 20101013-tp6c8np5w29su9pn4tedt8n5ks.jpg


Protein Gel 1:

Protein Gel 1
Protein Gel 1

Protein Gel 2:

Protein Gel 2
Protein Gel 2







Tuesday, October 5th, 2010: RNA Extraction and Protein Analysis, Part 1






2.EST386 subtracted libraries from oysters exposed to hypoxia Crassostrea gigas cDNA similar to glutathione peroxidase, mRNA sequence
3.EST128 subtracted libraries from triploid versus diploid in autumn Crassostrea gigas cDNA clone V-8 similar to putative glutathione peroxidase, mRNA sequence