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Extract DNA from gigas broodstock gonad tissue #277

Closed sr320 closed 6 years ago

yaaminiv commented 6 years ago

@laurahspencer and @kubu4 any input/advice on extracting DNA from histology cassettes?

yaaminiv commented 6 years ago

From conversations with @laurahspencer , it seems like the DNA extraction PAXgene Tissue DNA Kit did not work well because we don't have a shaker-incubator. I could try extractions with gigas, but without the shaker-incubator, is it worth it?

laurahspencer commented 6 years ago

Yes you can still do it- with some alterations I was able to successfully extract RNA.

I added glass beads to my samples during vortex/incubations. They are blue and in a small beaker on the lab bench in 209. I’d recommend autclaving again before use.

Alternative to incubator/ shaker:

There’s an incubator / shaker in the titrator room on the left when you walk in. It’s max RPM is not as high as the kit requires, so I’d recommend extending the time until you visually no longer see substantial undissolved tissue. Note: when you open this to load samples the temp quickly drops- you could set it higher than needed to warm before loading samples.

Another option for lower temp incubations:

On Wed, Jun 13, 2018 at 8:35 AM Yaamini Venkataraman < notifications@github.com> wrote:

From conversations with @laurahspencer https://github.com/laurahspencer , it seems like the DNA extraction PAXgene Tissue DNA Kit https://www.preanalytix.com/sites/default/files/handbooks/HB-0162-003-1080354-HB%20PAXgene%20Tissue%20DNA%201214%20WW.pdf did not work well because we don't have a shaker-incubator. I could try extractions with gigas, but without the shaker-incubator, is it worth it?

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yaaminiv commented 6 years ago

@laurahspencer What about extracting DNA for bisulfite sequencing?

sr320 commented 6 years ago

@yaaminiv what does the protocol say to do? we have a shaker incubator. For example what specific step in the protocol are you concerned about?

yaaminiv commented 6 years ago

@sr320 Okay I think I confused myself...

There are three different protocols to choose from:

  1. Purification of Genomic DNA from Sections of PAXgene Treated, Paraffin-Embedded Tissue
  2. Purification of Genomic DNA from PAXgene Treated Tissue Samples
  3. Purification of Genomic DNA from PAXgene Treated, Paraffin-Embedded Blocks of Tissue

I believe I need to use the third protocol.

In step 15, I need to incubate for an hour at 56ºC and 1400 rpm. @laurahspencer said our machine doesn't go this high, so I need to extend the incubation time to dissolve the issue.

In step 17, I need to incubate for an hour at 80ºC and 1400 rpm. I do not think our shaker-incubator gets to this temperature, which is why Laura needed to use the vortex/oven combination.

sr320 commented 6 years ago

I would boot up those incubators / shakers and confirm what they do and modify steps accordingly.

yaaminiv commented 6 years ago

The maximum temperature on the shaker-incubator is 60ºC. The maximum rpm is 500. For step 17, I would assume I need the vortex/oven combination.

sr320 commented 6 years ago

Ok

On Wed, Jun 13, 2018 at 10:45 AM Yaamini Venkataraman < notifications@github.com> wrote:

The maximum temperature on the shaker-incubator is 60ºC. The maximum rpm is 500. For step 17, I would assume I need the vortex/oven combination.

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yaaminiv commented 6 years ago

Update: @emmats has an Eppendorf Thermomixer that can go up to 3,000 rpm and 100°C, so I will try and use it for DNA extractions

yaaminiv commented 6 years ago

I will test the protocol using 1 sample pre-treatment tomorrow. I will use the Qubit to quantify DNA yield.

Assuming all goes well, I'm planning on extracting DNA from all my post-treatment samples (10 low pH and 10 ambient pH samples). I'm not planning on extracting any DNA from pre-treatment samples besides tomorrow's test sample. Am I forgetting to consider any factors in my plan?

yaaminiv commented 6 years ago

On hold...turns out the xylene we thought the Young lab had was actually xylene waste!

laurahspencer commented 6 years ago

I borrowed some xylene from the young lab, and I think there is some left in a large bottle underneath the hood (near the ethanol).

On Thu, Aug 2, 2018 at 5:26 AM Yaamini Venkataraman < notifications@github.com> wrote:

On hold...turns out the xylene we thought the Young lab had was actually xylene waste!

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yaaminiv commented 6 years ago

@laurahspencer That xylene is actually a xylene waste bottle.

sr320 commented 6 years ago

Ask Graham if he has any.

On Wed, Aug 1, 2018 at 12:35 PM Yaamini Venkataraman < notifications@github.com> wrote:

@laurahspencer https://github.com/laurahspencer That xylene is actually a xylene waste bottle.

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laurahspencer commented 6 years ago

Can you post a photo of the bottle you’re talking about?

On Thu, Aug 2, 2018 at 5:34 AM Yaamini Venkataraman < notifications@github.com> wrote:

@laurahspencer https://github.com/laurahspencer That xylene is actually a xylene waste bottle.

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laurahspencer commented 6 years ago

I’m wondering if I used waste xylene in my extractions ...

On Thu, Aug 2, 2018 at 5:39 AM Steven Roberts notifications@github.com wrote:

Ask Graham if he has any.

On Wed, Aug 1, 2018 at 12:35 PM Yaamini Venkataraman < notifications@github.com> wrote:

@laurahspencer https://github.com/laurahspencer That xylene is actually a xylene waste bottle.

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kubu4 commented 6 years ago

You didn't use xylene waste - they're out of xylene.

On Wed, Aug 1, 2018, 12:45 laurahspencer notifications@github.com wrote:

I’m wondering if I used waste xylene in my extractions ...

On Thu, Aug 2, 2018 at 5:39 AM Steven Roberts notifications@github.com wrote:

Ask Graham if he has any.

On Wed, Aug 1, 2018 at 12:35 PM Yaamini Venkataraman < notifications@github.com> wrote:

@laurahspencer https://github.com/laurahspencer That xylene is actually a xylene waste bottle.

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yaaminiv commented 6 years ago

@sr320 Sam checked MyChem and they're fully out of xylene. We're going to order some more

kubu4 commented 6 years ago

I checked Friedman Lab, not Young Lab. Don't have access to Young Lab MyChem inventory.

You should contact them directly to ask if we may use some xylene.

On Wed, Aug 1, 2018, 12:58 Yaamini Venkataraman notifications@github.com wrote:

@sr320 https://github.com/sr320 Sam checked MyChem and they're fully out of xylene. We're going to order some more

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yaaminiv commented 6 years ago

@kubu4 Ohhhh, okay. I will email Graham and see if they have more xylene I could use.

yaaminiv commented 6 years ago

Checked with Graham and Chris. Young lab is out of xylene. Created new issue for purchasing, so I'll resume extractions when we have the chemical.

yaaminiv commented 6 years ago

I tried the protocol twice (see here and here), and both times I had low yields (3.05 ng/µL and 1.16 ng/µL). I would need 130 ng/µL for MBD-Seq.

@laurahspencer What were your yields when you tried the protocol? How long did you vortex with glass beads? I've been vortexing for about 20 s and @kubu4 suggested vortexing for longer to try and mimic the conditions of a Tissue Lyser, but I wanted to see if you also tried that (only asking these questions because I don't see the information in your lab notebook!).

How many more times should I try before I try extracting DNA from a different tissue?

laurahspencer commented 6 years ago

My RNA extraction from fixed gonad was the most successful, and I vortexed with glass beads for way longer - 45 minutes

See this post for yield details: https://laurahspencer.github.io/LabNotebook/RNA-isolation/

On Wed, Aug 8, 2018 at 8:40 AM Yaamini Venkataraman < notifications@github.com> wrote:

I tried the protocol twice (see here https://yaaminiv.github.io/Gigas-Broodstock-DNA-Extraction-Part2/ and here https://yaaminiv.github.io/Gigas-Broodstock-DNA-Extraction-Part3/), and both times I had low yields (3 ng/µL and 1.16 ng/µL).

@laurahspencer https://github.com/laurahspencer What were your yields when you tried the protocol? How long did you vortex with glass beads? I've been vortexing for about 20 s and @kubu4 https://github.com/kubu4 suggested vortexing for longer to try and mimic the conditions of a Tissue Lyser, but I wanted to see if you also tried that (only asking these questions because I don't see the information in your lab notebook!).

How many more times should I try before I try extracting DNA from a different tissue?

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kubu4 commented 6 years ago

But what about your DNA extractions?

On Wed, Aug 8, 2018, 01:19 laurahspencer notifications@github.com wrote:

My RNA extraction from fixed gonad was the most successful, and I vortexed with glass beads for way longer - 45 minutes

See this post for yield details: https://laurahspencer.github.io/LabNotebook/RNA-isolation/

On Wed, Aug 8, 2018 at 8:40 AM Yaamini Venkataraman < notifications@github.com> wrote:

I tried the protocol twice (see here https://yaaminiv.github.io/Gigas-Broodstock-DNA-Extraction-Part2/ and here <https://yaaminiv.github.io/Gigas-Broodstock-DNA-Extraction-Part3/ ), and both times I had low yields (3 ng/µL and 1.16 ng/µL).

@laurahspencer https://github.com/laurahspencer What were your yields when you tried the protocol? How long did you vortex with glass beads? I've been vortexing for about 20 s and @kubu4 https://github.com/kubu4 suggested vortexing for longer to try and mimic the conditions of a Tissue Lyser, but I wanted to see if you also tried that (only asking these questions because I don't see the information in your lab notebook!).

How many more times should I try before I try extracting DNA from a different tissue?

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laurahspencer commented 6 years ago

The DNA extractions were not very successful, but I did not incubate with glass beads. For the 8 samples I yielded the following (ng/uL):

On Wed, Aug 8, 2018 at 10:27 PM, kubu4 notifications@github.com wrote:

But what about your DNA extractions?

On Wed, Aug 8, 2018, 01:19 laurahspencer notifications@github.com wrote:

My RNA extraction from fixed gonad was the most successful, and I vortexed with glass beads for way longer - 45 minutes

See this post for yield details: https://laurahspencer.github.io/LabNotebook/RNA-isolation/

On Wed, Aug 8, 2018 at 8:40 AM Yaamini Venkataraman < notifications@github.com> wrote:

I tried the protocol twice (see here https://yaaminiv.github.io/Gigas-Broodstock-DNA-Extraction-Part2/ and here <https://yaaminiv.github.io/Gigas-Broodstock-DNA- Extraction-Part3/ ), and both times I had low yields (3 ng/µL and 1.16 ng/µL).

@laurahspencer https://github.com/laurahspencer What were your yields when you tried the protocol? How long did you vortex with glass beads? I've been vortexing for about 20 s and @kubu4 https://github.com/kubu4 suggested vortexing for longer to try and mimic the conditions of a Tissue Lyser, but I wanted to see if you also tried that (only asking these questions because I don't see the information in your lab notebook!).

How many more times should I try before I try extracting DNA from a different tissue?

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kubu4 commented 6 years ago

What were your total yields?

On Wed, Aug 8, 2018, 07:19 laurahspencer notifications@github.com wrote:

The DNA extractions were not very successful, but I did not incubate with glass beads. For the 8 samples I yielded the following (ng/uL):

  • 2.480
  • 3.820
  • 0.808
  • 8.990
  • 0.567
  • 3.663
  • 1.353
  • 0.881

On Wed, Aug 8, 2018 at 10:27 PM, kubu4 notifications@github.com wrote:

But what about your DNA extractions?

On Wed, Aug 8, 2018, 01:19 laurahspencer notifications@github.com wrote:

My RNA extraction from fixed gonad was the most successful, and I vortexed with glass beads for way longer - 45 minutes

See this post for yield details: https://laurahspencer.github.io/LabNotebook/RNA-isolation/

On Wed, Aug 8, 2018 at 8:40 AM Yaamini Venkataraman < notifications@github.com> wrote:

I tried the protocol twice (see here https://yaaminiv.github.io/Gigas-Broodstock-DNA-Extraction-Part2/ and here <https://yaaminiv.github.io/Gigas-Broodstock-DNA- Extraction-Part3/ ), and both times I had low yields (3 ng/µL and 1.16 ng/µL).

@laurahspencer https://github.com/laurahspencer What were your yields when you tried the protocol? How long did you vortex with glass beads? I've been vortexing for about 20 s and @kubu4 https://github.com/kubu4 suggested vortexing for longer to try and mimic the conditions of a Tissue Lyser, but I wanted to see if you also tried that (only asking these questions because I don't see the information in your lab notebook!).

How many more times should I try before I try extracting DNA from a different tissue?

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laurahspencer commented 6 years ago

Total yields (ng):

On Thu, Aug 9, 2018 at 12:22 AM, kubu4 notifications@github.com wrote:

What were your total yields?

On Wed, Aug 8, 2018, 07:19 laurahspencer notifications@github.com wrote:

The DNA extractions were not very successful, but I did not incubate with glass beads. For the 8 samples I yielded the following (ng/uL):

  • 2.480
  • 3.820
  • 0.808
  • 8.990
  • 0.567
  • 3.663
  • 1.353
  • 0.881

On Wed, Aug 8, 2018 at 10:27 PM, kubu4 notifications@github.com wrote:

But what about your DNA extractions?

On Wed, Aug 8, 2018, 01:19 laurahspencer notifications@github.com wrote:

My RNA extraction from fixed gonad was the most successful, and I vortexed with glass beads for way longer - 45 minutes

See this post for yield details: https://laurahspencer.github.io/LabNotebook/RNA-isolation/

On Wed, Aug 8, 2018 at 8:40 AM Yaamini Venkataraman < notifications@github.com> wrote:

I tried the protocol twice (see here <https://yaaminiv.github.io/Gigas-Broodstock-DNA-Extraction-Part2/

and

here <https://yaaminiv.github.io/Gigas-Broodstock-DNA- Extraction-Part3/ ), and both times I had low yields (3 ng/µL and 1.16 ng/µL).

@laurahspencer https://github.com/laurahspencer What were your yields when you tried the protocol? How long did you vortex with glass beads? I've been vortexing for about 20 s and @kubu4 <https://github.com/kubu4

suggested vortexing for longer to try and mimic the conditions of a Tissue Lyser, but I wanted to see if you also tried that (only asking these questions because I don't see the information in your lab notebook!).

How many more times should I try before I try extracting DNA from a different tissue?

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kubu4 commented 6 years ago

Is there a notebook entry we can check out for these samples?

laurahspencer commented 6 years ago

Here is the entry for the first 4 samples - I don't have an online notebook entry for the last 4, but can do some digging if needed tomorrow.

https://laurahspencer.github.io/LabNotebook/Test-DNA-Extraction/

On Thu, Aug 9, 2018 at 12:33 AM, kubu4 notifications@github.com wrote:

Is there a notebook entry we can check out for these samples?

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yaaminiv commented 6 years ago

Ah, you used the Tissue Tearer instead of the glass beads + vortex combination I'm using. @kubu4 is it worth trying the protocol this way?

@laurahspencer Why did you add glass beads to all of your vortex/incubations? Wouldn't that risk shearing the DNA?

kubu4 commented 6 years ago

To clarify, @laurahspencer only used glass beads for RNA isolations. She used the TissueTearer for DNA isolation - no beads.

As we talked about yesterday, without having the TissueLyser from Qiagen, you'll have to empirically determine the best means to get good yields of intact DNA. @laurahspencer's methodology has decent yields, but still needs to be run on a gel to assess DNA integrity.

@yaaminiv, you can give the TissueTearer a shot, as well as increasing vortex times with glass beads to see what changes result in improved yields. You'll also need to evaluate the impacts of your various changes on DNA integrity by running samples on the Bioanalyzer and/or agarose gels.

yaaminiv commented 6 years ago

@laurahspencer How much Buffer TD5 did you use at the end? I don't see that detail in your lab notebook, and I know the protocol says you can use 50-200 µL. I've been using 50 µL of Buffer TD5, and I'm wondering if you did something different.

@kubu4 Tomorrow I can test the TissueTearer and increased vortex times (20 minutes and 45 minutes?), as well as run samples on the Bioanalyzer.

kubu4 commented 6 years ago

I wouldn't increase the vortex times so drastically to start (you've only done 20 seconds so far, right?). Maybe test 2, 5, and 10 minutes?

kubu4 commented 6 years ago

I would also increase the number of samples you use for testing. Currently, an n=1 is (potentially) not very informative. That particular tissue sample could be bad.

yaaminiv commented 6 years ago

I could do n = 2 or n = 3 per method?

kubu4 commented 6 years ago

n = 3 seems ok.

yaaminiv commented 6 years ago

Lab notebook

I tried four different try four different methods for lysating my samples (Steps 12-13):

Making these changes did not seem to help my extractions:

Tube Initial Mass (g) DNA Concentration (ng/µL)
T1-TT 0.0211 2.09
T1-V2 0.0210 1.72
T1-V5 0.0206 2.82
T1-V10 0.0208 0.524
T2-TT 0.0208 2.05
T2-V2 0.0204 N/A
T2-V5 0.0201 0.484
T2-V10 0.0204 0.804
T3-TT 0.0202 9.60
T3-V2 0.0205 6.20
T3-V5 0.0205 0.644
T3-V10 0.0202 2.92

I promised I would return the thermomixer first thing Monday morning. I could do some more trials tomorrow, but I'm not entirely sure if it's worth it. Should I move on to something different?

kubu4 commented 6 years ago

What were your yields? Concentrations don't tell us much and I can't seem to find the elution volume that you used in your notebook entry.

yaaminiv commented 6 years ago

@kubu4 I used an elution volume of 50 µL! Total amt. DNA (ng):

104.5 86 141 26.2 102.5 N/A 24.2 40.2 480 310 32.2 146

laurahspencer commented 6 years ago

@yaaminiv Regarding elution volume for my DNA attempts, I rinsed twice with 100 ul elution buffer (total volume 200ul) to try to maximize the yield.

For the RNA, I rinsed with 50ul since my test yields were good and my goal was a concentrated solution.

yaaminiv commented 6 years ago

I was able to extract DNA from test samples without using a thermomixer (see my lab notebook post)!

Two questions:

  1. Can I run samples on the Bioanalyzer this week so I can see if using the TissueTearor excessively sheared my DNA?
  2. During lab meeting we discussed doing bisulfite sequencing with a Qiagen kit that only needed 1 ng/µL since my yields are too low for MethylMiner. Is this still an option?
sr320 commented 6 years ago

Yes and yes

sr320 commented 6 years ago

seems like we have a plan .. see also https://github.com/RobertsLab/resources/issues/275#issuecomment-424408053 will close