Hello and welcome to Paul Ehlen's FISH 441 Notebook.
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Wednesday, November 26th, 2013

Ran qPCR again for 2 different samples (A2 before and after treatment) plus a water control. Used a lower annealing temperature. qPCR was again unsuccessful. It is likely that the hsp90 gene is not conserved across the anemone and that hsp sequences from the model anemone Nematostella vectensis are not applicable to Anthopleura elegantissima.

Tuesday, November 25th, 2013

Created cDNA for 8 samples as per lab protocol with minor changes in volumes.
Ran qPCR for 2 samples plus control to test primers. Used A1 (a control anemone) before and after treatment.
qPCR was unsuccessful.

Monday, November 24th, 2013

RNA extraction as per lab protocol.


Wednesday, November 20th, 2013

No treatment. Sampled.
Removed two pieces of tissue approximately 4 cu. mm. each from each of the four anemones. 8 samples total. Placed on dry ice immediately. Put into -80C freezer within 20 min.

Tuesday, November 19th, 2013

Administered treatment

Monday, November 18th, 2013

Administered treatment.

Sunday, November 17th, 2013

Danny administered treatment.

Saturday, November 16th, 2013

Jessica administered treatment.


Experimental Design: Sample Processing
8 tissue samples for hsp 90 gene expression level analysis.

Extract RNA
Create cDNA
Run qPCR

Experimental Design: Statistical Analysis
Hypothesis: Repeated and regular exposure to air and heat will triggered elevated levels of hsp 90 gene expression in the inter-tidal anemone, Anthopleura elegantissima.
Null Hypothesis: Repeated and regular exposure to air and heat will have no effect on levels of hsp 90 gene expression in the inter-tidal anemone, Anthopleura elegantissima.

T-Test on mean level of hsp 90 gene expression in control and treatment groups.


Friday, November 15th, 2013

Control tank 13C. One mangled anemone in nudibranch tank. One dead anemone floating in main holding, not an experimental animal. All anemones including experimental animals exhibiting tentacle bleaching, including 4 experimental anemones. 1/8-1/6 tentacles bleaching. Mouths intact and not gaping. All anemones appear normal otherwise. Have not yet fed anemones.

Administered treatment from 10:20 - 12:20. Achieved target temp of 23C immediately and maintained steady throughout. Anemone behavior during treatment similar to previous day. For the first time, removed lids from control animal vessels in main control tank during 2 hour treatment even though they were not being treated. This was presumed to be of benefit in terms of water exchange/aeration.

Jessica and Garret finished treatment and reacclimated animals. Jessica is treating Saturday and Danny the Wonder Child is treating Thursday. Instead of the heat lamps, Danny will be using just the intensity of his glare to warm the animals.


Thursday, November 14th, 2013

Control tank at steady 13C. Treated animals from 11:15-1:15. Initial difficulty maintaining treatment temperature. Hovered around 18 C for first 1/2 hour of 2 hour treatment. Added second heat lamp and got it to target temp of 23C almost immediately. Maintained between 22 and 24 C for remainder of treatment. Maintain 1/2 cm H2O in each treatment vessel. Anemones sag almost immediately upon exposure to air and move bulk of body mass lower along rock until tentacles and some column submerged. Foot stays in place on rock for the most part. Reacclimated over period of 1 minute.


Wednesday, November 13th, 2013

Experimental Design Refinements:

We further reduced the scope of our experiment to accommodate time and money limitations. We will work with 4 anemones and 4 urchins. 2 of each species will be subjected to the "heat shock" treatment of air exposure and heat for 2 hours each day and 2 of each species will remain in temperate seawater 24/7. Sampling will be done twice, at the beginning before any treatments and at the end of the week of treatments on the day after the last heat shock. We will then quantify mRNA via qPCR and hsp protein quantification via a Western Blot for each of the 8 study animals. 2 tissue samples from each study animal will be taken at each of the two samplings, immediately put onto dry ice and then into -80C freezer within 2 hours. Heat for the heat shock treatments will be provided by a white heat bulb positioned approximately 45 cm above the study animals. Temperature will be maintained between 22-23 C throughout 2 hours "heat shock" treatments. Animals will be maintained in 1/2-1 cm of seawater during treatments. Treated animals will be reacclimated to temperate seawater over 1-2 minutes before being reintroduced to temperate holding tank.

For my part of our experiment, I will be looking at hsp 90 gene expression via quantification of hsp 90 mRNA. mRNA will be extracted as per our lab procedures. Then cDNA will be created via qPCR. I'm still not exactly sure how mRNA is quantified but I know there is a way and I'll find out from Claire. I will be running the mRNA/qPCR procedure on 8 samples. Jessica will be running the same protocol on 8 urchin samples for a total of 16 samples for our group.

Today in the lab:

We took the first round of tissue samples followed by the first heat shock treatment. Regarding sampling the urchins, I did not participate but it seem to take some time to obtain any significant quantity of tissue. Regarding the anemones, 6-8 mm3 of tissue was taken from the edge of the oral disc or the side of the column. 2 tissue samples were taken per animal, 1 for mRNA and 1 for protein analysis. Samples were placed in flip top tubes and immediately put on dry ice. They were later transferred to the -80C freezer. The anemones looked pretty deflated and sad after the stress of the tissue extraction. If done again, would probably take tissue and allow a day of recuperation before beginning heat shock treatments. Due to time constraints, commenced heat shock same day. After approximately 1 hour of heat shock, noticed anemones moving lower on their rocks and looking very flat and oozy. Determined to add a small amount of water to each vessel with the goal of easing the stress caused by the tissue sampling. Will continue this protocol change for the duration of the experiment by adding 1/2-1 cm of water to each anemone vessel before treatment is commenced.

Fairly easy to maintain temperature of treatment. Ambient temperature in room was 20 C so raising that 3 degrees C and maintaining it was not difficult. Temperate water temp was 13C.

All 4 treated animals seemed to survive the 2 hour exposure. All maintained a glossy and "moist" appearance throughout. The urchins moved in their vessel. At one point the vessel tipped slightly causing the water to drift to one corner. The urchins congregated in that corner soon after. Vessel was righted. Anemone behavior involved moving lower on rocks, presumably pursuing water. By 1 1/2 hours (1 hour after adding water), the tentacles of the two anemones were able to touch the water. By treatment's end, the anemones were approximately 15% submerged. All treated animals were reacclimated to temperate seawater but gradually adding the colder water to their vessels over 1-2 minutes. Not a lengthy acclimation but similar to what they might experience with an incoming tide. Will repeat treatment protocol for 6 more days followed by another round of tissue sampling.





Tuesday, November 12th.

Finalized plans for experiment. Determined to just work on green urchins and aggregating anemones and look at hsp gene expression and hsp protein levels across those two species. Will begin sampling and treatments on Wednesday, November 13th. Sampling 3 times plus one. 3 times for the primary experiment and 1 time at the end for another mini experiment. We will sample tomorrow before any treatments and will then begin treatments of air exposure/heat. Treatments will be daily for 1/2 of sample set for each species and will consist of full exposure to air and temperatures in the neighborhood of 70 F via a heat lamp.

Sampling will be done 3 times for the primary experiment. once before any treatment. once at the 4 mark before that day's treatment and once on the last day before that day's treatment. Then, as an additional data set, we will expose all animals and then sample all.

Friday, November 8th.

Moved 16 anemones into individual flow-through containers. We plan to do initial tissue sampling and begin treatments early next week. We have all animals. We are still firming up our tissue sampling protocol to maximize our chances of get good RNA. We will likely do our initial tissue homogenization/lysing immediately upon extraction of the tissue sample. We are now considering looking at HSP expression via both RNA quantification and protein quantification. Myself and at least another member or two of our team are feeling a little overwhelmed by the complexity of the process as we lack experience.

Collecting Tuesday, November 5th.

We went to Golden Gardens and were able to collect about 10 small green urchins ranging from 3-6 cm in diameter.

Collecting Trip Friday, Nov. 1.


We went collecting at local puget sound beach and collected approximately 20 A. elegantissima, 16 Pisaster seastars and 5 shaggy mouse nudibranchs. These were acclimated and introduced to the aquarium system in FSH.

It is very noteworthy for our experiment that the animals were collected during a period where surface water temperatures in Puget Sound approximate air temperatures, about 45-50 F. This means that, though most of our animals experience daily air exposure (maybe not the nudis), they have not been experiencing any significant heat stress for a month or more. Thus they should be experiencing little if any heat shock priming. So, the animals we expose and warm will be the treatment. We will divide our animals evenly among treatment and control by species. We will sample tissue 3 times for each animal. Once before any treatment. once at the halfway mark of the treatment and once at the end of the treatment. We need to discuss our tissue analysis protocol more extensively with Claire and Professor.d We want to measure expression of HSP or related genes in treatment and control groups. We can try to measure the proteins themselves via Western Blot (I don't know exactly if this can be done) or we could extract and amplify mRNA associated with the proteins and then quantify it using qPCR. I think that would mean we would need to make cDNA but I'm not sure if that means you lose the ability to quantify your initial amounts of RNA which is the goal.




Lab 5: Experimental Design

We made several refinements to our experimental design on Tuesday in consultation with Claire and Professor Roberts. In particular, we determined to increase our sample size for each species with a goal of n=16. 8 to undergo the daily heat/exposure stress and 8 to remain submerged in temperate seawater. We confirmed with Professor Jensen that we could use the aquarium room in FSH to house our animals and run our experiments. This is particularly important because of the temperature stability of the system and the ability to control our experiment effectively. Our current plan is green urchins, an intertidal seastar species, a nudibranch species and Anthopleura elegantissima, the aggregating anemone. We made plans to go collecting on the upcoming Friday. We also briefly discussed primers and also measuring proteins with Professor Roberts. I have not yet designed primers and plan to talk with Claire asap.

Lab 4: Protein SDS/PAGE and Western blot, Gel Electrophoresis & qPCR data analysis





Part 1: Making an agarose gel (This was done for us ahead of time and these instructions are copied verbatim from the lab manual)

Supplies and Equipment:

AGAROSE GEL POURING PROCEDURE
  1. Weigh 2g of agarose and mix with 150mL 1x TAE in a 1L flask
  2. Microwave solution for ~ 3 minutes. Keep an eye on the solution so that it does not boil over. You want the solution to be clear - no precipitate and no bubbles.
  3. Cool solution (you should be able to touch the flask for a few seconds), then add 12uL ethidium bromide(EtBr). WARNING: EtBr is a carcinogen be sure to wear gloves and appropriately dispose tip waste.
  4. Mix thoroughly by swirling, then pour into gel tray.
  5. Add gel combs. Using a clean pipet tip, pop any bubbles that could get in the way of your PCR product.
  6. After gel is set, wrap in plastic wrap (label with your initials and date) and place gel in the fridge if not using immediately.


Part 2: Agarose Gel Electrophoresis


ELECTROPHORESIS PROCEDURE
  1. Placed gel in gel box and fill with 1x TAE buffer (to fully cover wells)
  2. Removed combs from wells
  3. Loaded 7uL 100bp ladder in far left lane
  4. Loaded 20uL of your PCR sample into the gel (retain the remaining vol at -20ºC)
  5. Ran gel at ~ 100V for ~ 1hr
  6. Visualized the gel on the UV transilluminator


Part 3: Protein Extraction and Analysis Part 2 - SDS - Polyacrylamide Gel Electorophoresis (SDS-PAGE)

Supplies and Reagents


SDS-PAGE PROTOCOL
Also see Manufacturers Protocol / Manual: Precise™ Protein Gels
  1. Begin boiling water on hot plate.
  2. In a fresh, 1.5mL SCREW CAP tube add 15uL of your protein stock and 15uL of 2X Reducing Sample Buffer. Return your protein stock to the box in the -20C freezer labeled protein samples.
  3. Mix sample by flicking. Briefly centrifuge (10s) to pool liquid in bottom of tube.
  4. Boil sample for 5 mins.
  5. While sample is boiling, observe assembly of gel box and gels. Rinse gel wells thoroughly as demonstrated.
  6. When sample is finished boiling, immediately centrifuge for 1min. to pool liquid.
  7. Slowly load your entire sample into the appropriate well using a gel loading tip.
  8. Put lid on gel box and plug electrodes into appropriate receptacles on the power supply.
  9. Turn power supply on and set voltage to 150V. Run for 45mins. CHECK YOUR AGAROSE GEL RESULTS. MAKE SURE EVERYTHING IS SET UP FOR WESTERN BLOT.
  10. Turn off power supply and disconnect gel box from power supply.
  11. Remove lid from gel box.
  12. Disengage the tension wedge.
  13. Remove gel from gel box.
  14. Carefully crack open cassette to expose gel.
  15. Trim wells at top of gel.
  16. Notch a designated corner of the gel to help you remember the correct orientation of the gel (i.e. which is the top/bottom of the gel, which is the right/left side(s) of the gel)
  17. Proceed to Western Blotting protocol.



Part 4: WesternBreeze Chromogenic Western Blot Immunodetection

Supplies and Reagents

Western Blot Protocol – Performed as a class
Soak the filter paper, membrane and gel in Tris-Glycine Transfer Buffer for 15 minutes.
  1. Assemble the blotting sandwich in the semi-dry blotting appartus:
    1. Anode (+++)
    2. filter paper
    3. membrane
    4. gel
    5. filter paper
    6. cathode (---)
  2. Transfer the blot for 30 minutes at 20V
  3. Remove the gel from the sandwich and rinse off adhering pieces of gel with transfer buffer.
  4. Wash membrane 2 times, for 5 minutes each, with 20 mL of pure water.
  5. Put the membrane in the plastic box and add 10 mL of Blocking Solution. Cover and incubate overnight on a rotary shaker set at 1 revolution/second.
  6. Your TA will do the rest of the steps. After class tomorrow you can come and see your results.
  7. Decant liquid.
  8. Rinse the membrane with 20 mL of water for 5 minutes, then decant. Repeat.
  9. Incubate the membrane in 10 mL of Primary Antibody Solution. Decant the solution.
  10. Rinse the membrane with 20 mL of Antibody Wash for 5 minutes, then decant. Repeat 3 times.
  11. Incubate the membrane in 10 mL of Secondary Antibody Solution for 30 minutes. Decant.
  12. Wash the membrane for 5 minutes with 20 mL of Antibody wash, then decant. Repeat 3 times.
  13. Rinse the membrane with 20 mL of pure water for 2 minutes, then decant. Repeat twice.
  14. Incubate the membrane in 5 mL of Chromogenic Substrate until a purple band appears. This will occur between 1-60 minutes after adding the Chromogenic Substrate.
  15. Dry the membrane on a clean piece of filter paper to the open air.
Results: My sample was in well #13. Saw a faint green band at about 200 bp.


LAB 3: Quantitative Polymerase Chain Reaction and Protein Extraction. Woo hoo!

Creation of cDNA: From Lab 2

At the end of Lab 2, we had time to begin Lab 3 by creating cDNA from our extracted RNA samples.

Methods:

Added the following to a 0.5 mL tube:

Incubated for 5 minutes at 70C

Added 14 uL Master Mix which contains buffer(s), NTPs (nucleotides), mmLV-RT (reverse transcriptase) and H2O. Gave to Claire for storage.

Purpose of Lab 3: Perform qPCR on our cDNA samples from lab 2, extract proteins from a tissue sample and finalize our experimental animals for our lab project.

Part 1: qPCR

Supplies and Equipment (verbatim from the lab)

• PCR Plates (white); optically clear caps

• 1.5 ml microfuge tubes (RNAse free)

• Nuclease Free water

• filter tips

• Opticon thermal cycler

• kim wipes

• 2x Immomix Master Mix

• SYTO-13 Dye

• microfuge tube racks

• ice buckets

• timers

• cDNA samples (student provided)


Methods

Note: The intention was to run a qPCR on our cDNA and a negative control. However, I put DNA into all 4 of my tubes. Thus, no controls. I will compare my results with controls of other students.

Preparation of master mix:

Recipe from lab:

For a 25μl reaction volume:

Component
 Volume
 Final Conc.
Master Mix (SsoFast EvaGreen supermix)
 12.5µL
 1x
upstream primer, 10μM
 0.5μl
 2.5μM
downstream primer, 10μM
 0.5μl
 2.5μM
Ultra Pure Water
 10.5uL
 NA

Notes: Prepared enough master mix for 5 reactions. The primers in the master mix were for expression of the HSP70 gene which codes for one of the chaperone proteins in the heat shock protein complex.

Methods:

PCR conditions:
1. 95°C for 10 minutes
2. 95°C for 15s
3. 55 °C for 15 s
4. 72°C for 15 s (+ plate read)
5. Return to step 2 39 more times
6. 95°C for 10s
7. Melt curve from 65°C to 95°C, at 0.5°C for 5s (+plate read)

Observations of the final qPCR data will be made next week.

Making an Agarose Gel (Taken verbatim from the lab. These were prepared for us ahead of time.)

Supplies and Equipment:
• Micropipettes (1-1000 μl)
• Sterile filter pipette tips (1-1000 μl)
• Tip waste jar
• 1L flask
• agarose
• 1X TAE
• Ethidium bromide
• Microwave
• Gel rigs
• Kimwipes
• Lab coat
• Safety glasses
• gloves

AGAROSE GEL POURING PROCEDURE

Part 2: Protein Extraction

Supplies and Reagents (verbatim from the lab)

Sample ID# 79 PAC

Methods:

Note: Normally our sample would have been weighed but the scale was broken. L


Part 3: Experiment Mock-Up

We spent the remainder of our lab time discuss our research projects and forming teams based on study animal. I am a proud member of Team Nematocyst! Our research animals will most likely be a local species of sea anemone. Perhaps Anthopleura elegantissima, the aggregating anemone. My interest is in nematocyst regeneration, associated proteins and evidence of changes in protein levels/gene expression in individuals under various environmental stressors. These may include lowered pH, elevated temperature or contaminants.


Lab 2: RNA Isolation, Part 2


Lab 2 is a continuation of Lab 1. We will use the RNA samples from Lab 1.

Purpose

Supplies and Reagents (verbatim from the lab)

Methods

* We did not quantitate the RNA as our TA said we could but did not need to. Nevertheless, here is the quantitization protocol from the lab instructions.
RNA QUANTIFICATION
NOTE: Always keep your RNA samples on ice!

Conclusions:
This lab was a study in lab recipe following and lab techniques. Even though our pellet wasn’t white, it was more of a grey, we did get a pellet. That’s really the only result we got since we did not quantitize.
Reflections:
All appeared to go well. It would be good to lay out the entire lab in a physical lab notebook so that the whole experience was kind of a fill-in-the-blanks. Even though the steps of the extraction and isolation techniques are not difficult, they are precise and much attention must be paid.





Lab 1: DNA isolation; initiate RNA isolation (Note: I was not in attendance for this lab. As per instructions from Professor Roberts, write-up includes a rewrite of the methods as well as a comparison of the DNA extraction technique with the technique we used in FISH 340)

Purpose

Safety Considerations!!!

(Copied verbatim from Lab Manual)


Methods:

RNA Extraction Part 1
Supplies and Reagents

RNA ISOLATION PROTOCOL
  1. The snap cap tube containing the tissue sample was labeled with the investigator’s initials and the date using a lab marker. The sample was stored on ice until it was time for homogenization.
  2. 500uL of TriReagent was added to the 1.5mL snap cap tube containing the tissue and then stored on ice.
  3. The tissue was carefully homogenized using a disposable pestle.
  4. After the sample was completely homogenized, an additional 500uL of TriReagent was added to the tube and the tube closed tightly.
  5. The sample was then Vortexed vigorously for 15s.
  6. The homogenized tissue was then labeled and stored at -80 C.

DNA Extraction: Part 2

Supplies and Reagents

DNazol Extraction Protocol (Adapted from MRC manual)
      1. Using a sterile pestle, the tissue sample was homogenized in 0.5 mL of DNazol in a 1.5 mL sterile microfuge tube. After the tissue was homogenized, 0.5 mL more of DNazol was added and mixed well.
      2. The sample was incubated for 5 minutes at room temperature.
      3. The sample was spun at 10,000 x g (room temp) for 10 minutes.
      4. The supernatant was transferred to a new, labeled tube.
      5. 0.5 mL of 100 % ethanol was added to the sample.
      6. The sample was mixed by inverting the tube 5-8 times.
      7. The sample was stored at room temperature for 1 minute.
      8. The DNA (a cloudy precipitate) was removed and put in a new tube using a pipette.
      9. The sample was allowed to sit at room temp for 1 minute and the rest of the lysate was removed (the liquid that was not DNA).
      10. The DNA was washed with 1 mL of 75% ethanol. The ethanol was pipetted into the DNA tube which was then inverted 6 times and allowed to sit for 1 minute. The ethanol was then removed from the tube and the process repeated.
      11. Any remaining ethanol left at the bottom of the tube after the second wash was removed with a small pipette.
      12. 300 µL of 0.1% DEPC water was added to the DNA and pipetted up and down multiple times to dissolve.
      13. The DNA sample was then brought to the Nanodrop to quantify.
DNA Quantification
        1. 2µL of 0.1%DEPC-H20 was pipetted onto the Nanodrop pedestal and the arm lowered.
        2. "dsDNA" was selected from the pulldown menu
        3. "Blank" was clicked to zero the instrument.
        4. "Measure" was clicked and the DNA concentration (ng/µL), A260/280 ratio and A260/230 ratio recorded. NOTE: The Nanodrop uses the Beer-Lambert Law to calculate DNA concentration.
        5. The arm was raised and the sample wiped off with a KimWipe
        6. The stock DNA sample was labeled with the word "DNA", source organism/tissue, investigator’s initials, date and the concentration in ug/uL.
        7. The sample was stored at -20ºC.b

Comparison with DNA Extraction in FISH 340 Lab

The method used for DNA extraction in the 441 lab uses the DNAzol reagent from Life Technologies whereas the method for 340 utilized the DNeasy kit. Both methods lysed the cell to liberate the DNA, removed cellular debris and precipitated out the DNA. The 441 method also suspended the DNA in solution as well as moving on to quantify the DNA.
Whereas the 441 method ended with the DNA suspended in NaOH, the 340 method ended with the DNA affixed to a synthetic membrane. The 441 method used a pestle to homogenize the sample whereas the 340 method used vortexing. For the 441 method, the lysing procedure was incubated at room temperature whereas the 340 method incubated at 56 C during this phase.
In the 441 method, the cell is lysed and the DNA isolated in a single step with the DNAzol reagent whereas in the 340 method the cell is lysed in the first step with buffer ATL and the DNA isolated later on the membrane in a second step with buffer AL and ethanol.
Both processes seem roughly equal in terms of complexity, number of steps and number of reagents. It is not known by this investigator which of the methods yields a more usable and reliable sample. Nor is it known which method is more costly. The 340 method is more time consuming requiring about 3 hours to complete whereas the 441 method may be completed in less than ½ hour.