RobertsLab
Extract DNA and RNA from 6 cod blood samples
Cod blood samples for this test DNA + RNA extraction. I've provided 8 (4 each for treatment, 5C and 9C) in case any of these samples are missing or have very little blood.
| Temperature | Sample # |
|---|---|
| 5 | 129 |
| 5 | 149 |
| 5 | 154 |
| 5 | 155 |
| 9 | 84 |
| 9 | 93 |
| 9 | 111 |
| 9 | 114 |
Alrighty, this is what we're looking at.
Here's notebook entry:
https://robertslab.github.io/sams-notebook/posts/2024/2024-08-29-DNA-and-RNA-Isolations---Gadus-macrocephalus-Blood/
Elution volumes were 15uL for DNA and 15uL for RNA.
- DNA Quantification (Google Sheet)
| Sample_ID | Concentration(ng/uL) |
|---|---|
| 84 | 12.5 |
| 93 | 3.42 |
| 111 | 18.3 |
| 114 | 58.8 |
| 129 | 0 |
| 149 | 20.8 |
| 154 | 7.76 |
- RNA Quantification (Google Sheet)
| Sample_ID | Concentration(ng/uL) |
|---|---|
| 84 | 18.6 |
| 93 | 28 |
| 111 | 22.8 |
| 114 | 20.6 |
| 129 | 7.9 |
| 149 | 22.2 |
| 154 | 21.8 |
I couldn't locate sample 155, BTW.
I think isolations could've been better. The blood coagulated into a dense goo as soon as I added the lysis buffer/proteinase k and didn't ever really break down. I probably should've tried to physically homogenize the samples after they lysis step showed minimal impact...
Azenta's mimimum requirements:
Strand-specific RNA-seq: 500ng Ultra-low input RNA-seq: 20ng
WGBS: 500ng
Based on those numbers, we are short on all samples for Strand-specific RNA-seq and WGBS.
However, we could do the ultra-low input RNA-seq.
Additionally, there are many WGBS library kits that handle low input amounts of DNA (i.e. which we could process all of the DNA samples listed above).
lets move forward with 16C and 9C cod blood samples getting DNA and RNA from samples... @laurahspencer can you advise on sample IDs, locations
I'd like to move forward with extracting RNA & DNA from all blood samples (four temperature treatments, 0C, 5C, 9C, and 16C). Max number of samples per temperature is n=40. If we need to reduce the number, we can omit the 5C temperature altogether.
I'll be on campus this Thursday (2/13) mid-morning and will inventory and move all these blood samples into their own box in the -80 for easy locating.
Is the goal to get RNA and DNA from the same sample? Or, are there multiples of each sample, so that RNA can be taken from one and DNA from the other?
Spreadsheet of inventory of selected samples would be killer. Will allow me to determine what supplies we might need to order.
From same samples.
On Tue, Feb 11, 2025 at 3:20 PM kubu4 @.***> wrote:
Is the goal to get RNA and DNA from the same sample? Or, are there multiples of each sample, so that RNA can be taken from one and DNA from the other?
Spreadsheet of inventory of selected samples would be killer. Will allow me to determine what supplies we might need to order.
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But ballpark is 200?
On Tue, Feb 11, 2025 at 3:29 PM Laura H Spencer @.***> wrote:
I'll inventory on Thursday so we ensure accurate info!
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@sr320 - Potentially good project for that NOAA person?
Or, do you want to stick with handling it "in-house?" 😃
Lets go ahead and extract these in house - and will need to make this a priority given funding deadline.
@laurahspencer - Did you happen to get a spreadsheet put together for these on Thursday? Also, did you happen to consolidate these into their own boxes? If yes, where might I find them?
Yes, below is the spreadsheet link! First tab is all blood samples in -80 that are available for DNA/RNA extractions. We actually have 131 samples, couple more than I thought. I forgot to move them to their own box, sorry! The spreadsheet identifies which box they are in (and where in -80). Second tab of spreadsheet is keeping track of what happened to other blood samples. Third has photos inside sample boxes. You'll notice most blood samples ("# B") have a faint pink color.
I'm available next week to help if needed! Thursday is good between 10am-4pm but I could make any day work.
https://docs.google.com/spreadsheets/d/1xf3rVOlvoYX6A9UeCAU4MhHb6e1dTR0YATeaFV3Jb_M/edit?usp=sharing
I think the extraction kits will be arriving tomorrow (finally!), so I'll get this started when they arrive.
However, I'll be out of town for a week as of next Tuesday. So, if we want to keep this moving, @laurahspencer I'll need you step in then to keep extracting samples. I'll keep everyone updated on status until then. Just wanted to give people the heads up/reminder about my upcoming time off.
Looks like extraction kits are shipping via FedEx ground, so won't be here until Thursday...
I have the kits, so am going to try to get some samples extracted today, but then I'm out of a week.
@kubu4 - awesome, that sounds like a good plan. You're the paired rna/dna extraction pro, so I think it makes sense to wait until you're back to continue with the extractions so I don't mess them up! If you would like an assistant when you return please let me know your lab schedule and I'll be there!
Hmm, well, like before, isolations didn't really go particularly great:
DNA Quantification
| Sample | Concentration (ng/uL) | Volume (uL) | Yield (ng) |
|---|---|---|---|
| 37B | 5.22 | 40 | 208.8 |
| 38B | 1.29 | 40 | 51.6 |
| 40B | Out of range | 40 | 0 |
| 41B | Out of range | 40 | 0 |
| 42B | 1.73 | 40 | 69.2 |
| 43B | 0.46 | 40 | 18.4 |
RNA Quantification
| Sample | Concentration (ng/uL) | Volume (uL) | Yield (ng) |
|---|---|---|---|
| 37B | 9.76 | 40 | 390.4 |
| 38B | Out of range | 40 | 0 |
| 40B | Out of range | 40 | 0 |
| 41B | Out of range | 40 | 0 |
| 42B | 17.5 | 40 | 700 |
| 43B | 6.82 | 40 | 272.8 |
Serendipitously, I happened to be talking to @mgavery yesterday and she mentioned difficulties working with fish blood (specifically) and indicated she had a protocol that they've optimized. @mgavery - When you have a sec, could you please upload that here?
Anyway, fingers crossed her protocol works a bit better, as our current approach is not great, to say the least...
You're gonna love this! Good old fashioned lab notebook copy :). This is the modified protocol using Qiagen's DNeasy blood and tissue kit.
Important info about the sample - the blood was collected in hepranized syringes, centrifuged to collect RBC layer and then frozen. It's not whole blood. ..but I do think the "goo" bit was why the protocol was modified
It's not whole blood
Dang, hopefully the "goo" bit will help over here.
Thanks!!!
@laurahspencer - I'm going to try another homegenization effort next week using @emmats her "Bullet Blender" to see if this will add in disrupting the coagulation (which, at this point, is the most likely reason for poor yields).
However, if this doesn't pan out either, you may want to start thinking about a different approach to this project (i.e. different tissues)? @sr320 has indicated that there's a bit of a time crunch, so you should potentially consider alternatives.
Yay! Bullet Blender worked great!
Recovered _micro_grams of DNA (which we would expect for a successful isolation) from virtually all samples.
Similarly, recovered nearly 1ug of RNA from many of the samples.
I've submitted an order for one of our own Bullet Blenders, but in the meantime, I'll start coordinating with Emma to use hers and move things forward.
Notebook:
https://robertslab.github.io/sams-notebook/posts/2025/2025-04-02-DNA-RNA-Isolation-and-Quantification---G.macrocephalus-Blood-Using-Bullet-Blender-and-Zymo-Quick-DNA-RNA-MiniPrep-Plut-Kit/index.html
DNA
Qubit (Google Sheet):
| sample_ID | Concentration(ng/uL) | Volume(uL) | Dilution^[A dilution value of 1 means the sample was not diluted.] |
Total(ng) |
|---|---|---|---|---|
| 82B | 9.66 | 40 | 1 | 386.4 |
| 83B | Out of range | 40 | 1 | #VALUE! |
| 86B | Out of range | 40 | 1 | #VALUE! |
| 88B | 55.8 | 40 | 1 | 2232 |
| 89B | 54.2 | 40 | 1 | 2168 |
| 90B | 67.6 | 40 | 1 | 2704 |
| 83B-1:5-dilution | 40.2 | 40 | 5 | 8040 |
| 86B-1:5-dilution | 60.2 | 40 | 5 | 12040 |
RNA
Qubit (Google Sheet):
| sample_ID | Concentration(ng/uL) | Volume(uL) | Total(ng) |
|---|---|---|---|
| 82B | 5.7 | 40 | 228 |
| 83B | 42 | 40 | 1680 |
| 86B | 47.2 | 40 | 1888 |
| 88B | 24.8 | 40 | 992 |
| 89B | 19.7 | 40 | 788 |
| 90B | 22.4 | 40 | 896 |
Just providing an update.
Extracted 12 more samples last week - went well.
No activity this week, as Emma is unavailable to provide access to her lab, and SAFS still hasn't submitted our order for the Bullet Blender machine that we've been using in Emma's lab.