Evan’s Capstone – eNotebook 7

Monday 3/21/2016 – Test qPCR Results

The results of the initial test run of the qPCR conducted last week for the Actin and CYP1A genes came back after using the same procedure posted up in the previous notebook. Both genes single-plexed came back with good amplification at the cycle timing that we were looking for.

 

In addition, the control wells did not amplify as expected indicating the DNAsing procedure and Reverse-transcription was completed correctly for these samples, and hopefully the rest of them.

Test-Controls

Control Wells

However, the duplexed gene wells did not work correctly, with only the Actin wells significantly amplifying during the course of the qPCR.

Test-Duplex

Duplex, Actin in Green, CYP1A in Blue

There’s no guarantee as to what might have caused the duplexing to fail, whether it could have been a simple saturation of the DNA polymerase with Actin or interaction between the dyes I’m not sure (this is what testing plates are for!)

My plan for this week is to work on finishing up Reverse-transcribing the samples and get input on how to proceed with the qPCR. There’s the option of attempting to figure out the issue with the duplexing and correcting it if possible and optimizing that; or simply proceeding with single-plexing the gene targets. The latter might require a bit more work but will definitely get results if it goes as smoothly as this test run.

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Evan’s Capstone – eNotebook 6

Saturday 3/12/2016 – Big Update

DNAsing has been finished for every sample without problems following the same procedure as found in the Common Lab Protocols, with the only deviation being 5 minutes total of DNAse inactivation as compared to the 2 minutes stated there. The next step of the project is going to be getting ready to reverse transcribe the samples I DNAsed and then getting the information necessary to qPCR it all! To sum it up…

Remaining qPCR Steps:

  1. Reverse Transcribe 6 DNAsed samples (following lab standard protocol) – Complete
  2. Run test plate to check primer/probes, multiplexing, and reverse transcription
  3. Reverse Transcribe remaining 30 samples
  4. Run the qPCR for B-actin and CYP1A on all 36 samples
  5. Repeat steps 2 and 4 for MTA and VTG gene if time allows (using B-actin as the reference)

Test Sample Run of qPCR

Setup

1. Thaw Master Mix and nuclease-free water at room temperature and then vortex for 5 seconds.

  1. Number of reactions is 81 6, additional reactions will make it 83 7 total reactions to ensure against pipetting error.
  2. Setup will be 10 uL of Master Mix, 1 uL each of Forward and Reverse Primer in addition to Hydrolysis Probe(s), 4 uL of Template DNA (depending on concentration of reverse transcription products), and 3 uL of Nuclease-Free Water for each reaction (also depending)
  3. Total for test run: 70uL of Master Mix, 7 uL of F/R Primers+Probe(s), 21 uL of Nuclease-Free Water with 16 uL of mix added to each well after being vortexed briefly to mix.
  1. Add 4 uL of diluted Template DNA (1 ug/ul, maybe 10 ng/ul?) to each well from the respective samples, seal the optical plate, and centrifuge briefly to collect components at bottom of the wells.
  2. No Template Controls will receive an additional 4 uL of Nuclease-Free Water, No RT Controls will receive uL of non-RT “RNA” Sample, No Amp. Controls will receive an additional 10 uL of Nuclease-Free Water to replace DNA polymerase found in Master Mix.

(Standard) Thermal Cycling Conditions

1 Cycle of: 95 Celsius for 2 minutes to activate GoTaq

40 Cycles of: 95 Celsius Denaturation for 15 seconds followed by 60 Celsius Annealing/Extension for 1 minuteWellPlates

This data is going to subject to change however and will be updated as necessary until it is ready to be ran through. I’m keeping a full data set table which includes all the data gathered from the samples up to this date here, Capstone Sample Data. Plan is tentatively going to be to discuss and make changes to the procedure early next week and then hopefully run the test to determine if multiplexing is going to be possible later next week.

Evan’s Capstone – eNotebook 5

Wednesday 2/10/2016 and Thursday 2/11/2016

            Double update today since I didn’t have time to write up my report yesterday night and figured it would be simple to fill it in here! Wednesday was relatively simple, just finished up the final 7 RNA Isolations while today I worked on learning how to conduct proper DNAsing of the samples to prepare for reverse transcription and then qPCR.

Wednesday’s RNA Isolations:

3BRC and 9BRC (Rock Creek) in addition to 7BCO and 17BCO (Coulter Creek) were pulled at 12:20 PM to be used as the first set. The second set consisted of 1BRC and 12BRC with 19BCO and were pulled at 12:55PM. Samples were returned to the -80 freezer at 4:05 PM.

Only one slight deviation from the standard procedure that day and it was that Set 1 rested on ice after step 15 for 10 minutes while waiting for Set 2 in the centrifuge.

Nanodrop Results:

Example: X-B-SampleSite – 260/280: ###, 260/230: ###, Concentration ### ng/uL
1BRC – 1.82, 1.01, 436.4
3BRC – 1.95, 2.20, 1393.7
9BRC – 1.97, 2.14, 1279.0
12BRC – 1.91, 2.20, 872.8
7BCO – 1.92, 2.05, 611.0
17BCO – 1.92, 2.08, 766.1
19BCO – 1.91, 2.28, 925.0

Thursday’s DNAsing:

Relatively simple to learn, it went smoothly as far as I can tell other than some of the samples having lower post-DNAse nanodrops than before. The procedure was the same as what is listed in the Common Lab Protocols except on the step where on step where you add the deactivation reagent and incubate it, we let it incubate for 5 minutes as per the manufacturer procedure.

The first set of isolated samples was pulled at 12:05 PM and consisted of 3BMA and 5BMA in addition to 13BHA and 14BHA. As a note, 14BHA was the sample that only had 25 uL total of isolated sample because of a small liver sample size. This led me to adapt a procedure and dilute it down to 40 ng/uL as compared to the normal 200 ng/uL for every other sample concentration since its initial concentration was only 180 (with only about 20 uL when it calls for about 44). The second set of samples consisted of 6BRC and 7BRC (Rock Creek) with 11BSW and 13BSW (Swamp Creek) and were pulled at 2:05 PM.

Nanodrop Results Post-DNAse (same format):
3BMA – 1.97, 1.47, 109.6
5BMA – 1.96, 1.32, 125.8
13BHA – 2.01, 1.75, 111.1
14BHA – 1.89, 1.04, 28.3
6BRC – 1.97, 1.68, 107.8
7BRC – 1.99, 1.60, 111.8
11BSW – 1.91, 1.43, 113.5
13BSW – 1.98, 1.55, 130.3

Samples were returned to the -80 freezer in a new storage container (to signify they were DNAsed) at 3:55 PM for Set 1 and 5:30 PM for Set 2. It appears that a lot of concentration (and 260/230 values) were lost in the process of DNAsing, given that the 260/230 numbers normally decrease with concentration this was expected and given how little actual RNA is in 14BHA I am not surprised it only has a value of 1.04. The other samples were okay, was really wanting more in the 1.60 range but the 1.4+’s are okay too. Most of the samples lost a lot of concentration to DNAsing which was also expected since liver tissue samples are normally pretty high in DNA and RNA and then by removing the DNA it would make sense for the concentration to dip down more than other types of samples. Overall a good learning experience and the samples are likely still viable. Next day in lab will likely be better and I should be able to get all the other samples done if I work efficiently!

 

Evan’s Capstone – eNotebook 4

Thursday – 2/4/2016
            Back in the lab after calling in sick on Tuesday, have to make up time tomorrow! Just continuing the RNA isolations, should be done with the last 7 tomorrow (leaving me with a total of 35 samples between 6 watersheds).

Samples ran today was the last set to finish off Harris and Issaquah from Thursday (2 and 19 from Issaquah in addition to 11 and 16 from Harris) and those were pulled at 12:45 PM. The other set of samples was the beginning of Coulter Creek and Rock Creek (9BCO, 14BCO, 6BRC, and 7BRC) and were pulled at 1:30 PM.

Surprisingly, there was actually no deviations today at all from the common procedure as all the livers were of a large enough size in addition to having the lab to myself. The optimal strategy seems to be to start homogenization of the 2nd set while the 1st set is incubating for the first centrifuge step. It ends up leaving you swapping out samples constantly with there always being one set of samples in the centrifuge at any point in time!

Nanodrop Results for the Day:

2BIS – 260/280: 1.94, 260/230: 2.19, Concentration: 769.1 ng/ul
19BIS – 1.80, 2.12, 435.5
11BHA – 1.98, 2.19, 1265.6
16 BHA – 2.00, 2.11, 1473.3

6BRC – 1.97, 2.18, 1276.7
7BRC – 1.96, 2.21, 1860.0
9BCO – 1.92, 2.21, 825.7
14BCO – 1.77, 2.03, 446.5

I also seems like I finally figured out the Nanodrop machine, getting results on the first attempt for all but the Coulter Creek samples which was giving me errors. I later learned (Thanks Sam!) that the machine seems to respond well to 2 ul samples instead of 1 ul samples, as it seems to prevent column breakage which I think is what has been giving me such a hard time for the last 3 tries at the machine. Gives me a little more hope for the samples from last Thursday that maybe I simply needed to increase the water drop size for the machine. Would have liked better results from 19BIS and 14BCO, but they are pretty close and the concentrations are lower than the other samples so it is likely fine.

All samples were placed back in the -80 freezer at 5:10 PM and I’m going to try to work in some time tomorrow to get the last 7 samples done to finish off the week!

Evan’s Capstone – eNotebook 3

Thursday – 1/28/2016

Plan for today changed slightly, just continuing more RNA isolations in the hopes of getting them all done by next week and continuing on to the next part of the project. Operation running smoothly now, essentially had no downtime during sample work with the centrifuge running with either set of samples at a time. Also started work on two new creeks now that the first 6 samples of Swamp Creek and May Creek are finished!

First set of samples consisted of 11 and 13 of Issaquah (#BIS) in addition to 13 and 14 of Harris (#BHA) and were pulled at 11:50 AM. The second set was 12 and 15 of Harris with 3 and 17 of Issaquah which were pulled at 12:10 PM.

A few more deviations this time due to balancing centrifuge time with my supervisor; Set 1 rested for 4 additional minutes after Step 18 and then after Step 21 rested for longer than anticipated (8 minutes) and was re-centrifuged for 3 minutes to ensure integrity of pellet. Set 2 rested after Step 13 for 4 additional minutes and also rested after Step 18 for 2 additional minutes. Sample 14BHA ended up being re-suspended in 25 ul of DEPC-treated water instead of the usual 50 because of a small liver sample and subsequently a small pellet size.

Nanodrop results for the day:

12BHA – 260/280: 1.97, 260/230: 2.01, Concentration 720.6
13BHA – 1.98, 2.26, 1574.7
14BHA – 1.74, 2.08, 391.0 (~)
15BHA – 1.94, 2.09, 774.5

3BIS – 1.95, 2.09, 844.6
11BIS – 1.96, 1.90, 644.8
13BIS – 1.93, 2.27, 977.6
17BIS – 1.82, 1.57, 241.1 (~)

The tilde is to represent the two samples I am questioning on using given the less than optimal numbers (both have numbers lower than 1.8 which is what I’ve been looking for). In hindsight, 17BIS had a small liver sample size similar to 14BHA and given how low the concentration is, it probably would have been smart to limit the re-suspension to 25 ul for it also. I’m not sure if the small liver sample size is to blame for the low Nanodrop numbers? I will need to consult with someone about what to do with them.

All samples were placed in the -80 freezer at 4:40 PM following the Nanodrop results. My goal for next week will be to get at least 6 samples from each creek finished so that they can be brought through the next steps and start to see some results. May or may not have to pull an extra sample for Harris and Issaquah given the two “maybes” from today; We’ll see!

#rna, #salmon

Evan’s Capstone – eNotebook 2

Tuesday – 1/26/2016

Coho Salmon and Cutthroat Trout Liver Analyses

Continuing work on RNA isolations today, managed to double up on the samples run per day and actually ended up easier to do than Thursday’s due to running homogenization during incubation steps.

Samples were pulled from Swamp Creek and May Creek again, Set 1 being samples 2May, 6May, 9Swamp, 14Swamp at 11:36 AM; Set 2 consisted of 1May, 7May, 6Swamp, and 18Swamp and were pulled at 12:54 PM.

Samples were ran through RNA Isolation as indicated in the Common Lab Protocols like last Thursday’s samples (found at https://github.com/sr320/LabDocs/wiki/Common-Lab-Protocols).

Some more minor deviations due to timing between sets, less than before given overlap went pretty well overall during incubation times. The 1st set of samples were kept on ice from 2:30 PM to 3:30 PM until time was available to do the Nanodrop. The 2nd set of samples had to sit at Step 13 for 2 extra minutes, and on Step 10 for 3 minutes, then before Step 20 they had to remain in the isopropanol for 10 minutes while waiting for Nanodrops for the 1st set.

Nanodrop samples came back better than before (likely due to being more comfortable with the machine) and were conducted the same way as Thursday’s samples.

Set 1 ~
2BMA – 260/280: 1.99; 260/230: 2.14; Concentration: 1466.4
6BMA – 1.84, 1.87, 422.4
9BSW – 1.98, 2.12, 1505.1
14BSW – 1.98, 1.86, 1387.5

Set 2 ~
1BMA – 1.96, 1.82, 889.9
7BMA – 2.00, 2.10, 1841.1
6BSW – 1.98, 2.14, 1410.4
18BSW – 1.99, 1.82, 1699.6

Samples in Set 1 were placed in the -80 Celsius freezer at 3:50 PM while the Set 2 samples were in the freezer by 4:50 PM.

Seems to be even better than last week, plan for Thursday is to learn the process of DNasing the RNA samples and likely more RNA isolations!

Evan’s Capstone – eNotebook 1

Thursday – 1/21/2016

First day of Lab work on samples! Excited to get started on the study sites for the capstone project.

Samples from Swamp Creek (11 and 13) and May Creek (3 and 5) were pulled at 12:50 to be taken through the process of RNA Isolation as outlined in the Common Lab Protocols.

Some minor deviations due to conducting two different “sets” of two samples at a time and some overlaps when using the centrifuge with another lab member. Maybe try to figure out a better method of conducting multiple samples, maybe sets of 4 and then during the first incubation/centrifuging step set up another set of 4 samples?

During the first incubation of 15 minutes the second set of samples (5 and 13) incubated for 19 minutes while the first set of samples were in the centrifuge. After the 750 ul supernatant was pulled off and placed in a new snap-cap tube these two samples also rested for 5 minutes while waiting on the final ethanol washing step for the first set of samples (3 and 11). The first set of samples also had to be set to rest for 12 minutes before the final ethanol centrifuge, the ethanol supernatant was pulled off and the sample was set capped to ensure the ethanol did not evaporate off completely before the second 400 ul of ethanol could be added and the final washing step could be conducted.

The first set of samples were capped and held in the normal refrigerator until the second set was finished so we could bring them to the Nanodrop machine.

We set up the machine by first cleaning off the upper and lower lenses, placing 1 ul of clean water and initializing the nucleic acid option then cleaning the lenses and repeating the step again choosing the blank option. The lenses were cleaned in between each 1 ul sample and using the “measure” option.

Some errors occurred with new use of the machine (lower values or higher concentrations than expected, air bubbles in some situations, and some material not fully suspended on another sample), however the values from the successful runs of the machine were collected:

(ex. 3BMA is 3rd Sample, B for Cutthroat, MA for May Creek, SW for Swamp Creek)
3BMA – 260/280: 1.99; 260/230: 1.92; Concentration ng/ul: 1291.1
5BMA – 1.97, 1.80, 1111.3
11BSW – 1.96, 1.80, 745.7
13BSW – 1.98, 1.92, 1221.1

Completed samples placed back into the container and stored in -80 Celsius freezer at 5:15 PM and work concluded.

Good results and data collected so far and ready for work on Tuesday!

#rna, #salmon