West Coast Green Crab Experiment Part 62

Continuing RNA extractions

Methods

Prepare for extractions:

  1. Thoroughly clean RNA room, bench, and hood. Clean any equipment that will be used.
  2. Remove samples from the -20ºC and allow to thaw on the bench.
  3. Label 1 low-adhesion tube for each sample and an additional tube for the extraction blank.

Tissue processing:

  1. Remove crab heart from RNALater tube using an unfiltered pipet tip and place into a cleaned weigh boat. Cut a subsection of the tissue using a clean scalpel. Blot RNALater from tissue on a clean kim wipe folded in half. Use the pipet tip to place the subsection into the labelled tube. Repeat for all samples.
  2. Break up tissue samples using a disposable pestle. Once the texture is similar to wet sand, leave the pestle in the tube and proceed to the next sample.
  3. In the hood, add 500 µL of TRIzol. Mix using the disposable pestle already in the tube. Remove the pestle and ensure no sample is on it before disposing in a Ziploc bag.
  4. Add an additional 500 µL of TRIzol and pipet repeatedly to completely homogenize. Any remaining chunks of tissue should easily pass through the pipet tip.
  5. Let sit 5 minutes at room temperature. While samples are sitting, pre-cool the centrifuge to 4ºC.

Phase separation:

  1. Add 100 µl BCP and shake vigorously for 15 seconds.
  2. Let sit for 15 min at room temperature.
  3. Centrifuge 15 min at 4°C / 12,000 xg.
  4. While centrifuge is running, remove the glycogen from the -20ºC to thaw. Label 1 low-adhesion tube for each sample and blank.
  5. Transfer the upper, clear aqueous phase to a fresh tube (about 450 µL - 470 µL).

Pelleting RNA:

  1. Add 1 µL glycogen (5-20 mg / mL) and pipet to mix.
  2. Add 500 µl isopropanol and vortex gently.
  3. Incubate samples at -20ºC overnight. Samples can be incubated for up to 7 days at -20ºC.

Cleaning and resuspending RNA:

  1. Centrifuge 8 min at 4°C / 12,000 xg.
  2. While samples are centrifuging, prepare just enough 75% ethanol for samples (2000 µL).
  3. Remove and discard the supernatant, being careful not to disturb the pellet.
  4. Add 1000 µL 75% ethanol and vortex to mix.
  5. Centrifuge 5 min at 4°C / 7,500 xg.
  6. Remove and discard the supernatant, being careful not to disturb the pellet.
  7. Add 1000 µL 75% ethanol and vortex to mix.
  8. Centrifuge 5 min at 4°C / 7,500 xg. While samples are centrifuging, obtain all Qubit reagents and let them come to room temperature. Additionally, aliquot RNAse-free water and place in a heat block set to 56ºC.
  9. Remove and discard the supernatant, being careful not to disturb the pellet. For this final step, it helps to remove supernatant with the 1000 µL pipet, then go back in with a 20 µL pipet to remove the remainder of the liquid.
  10. Air-dry the RNA pellet for 3 min. After 3 min, remove any remaining liquid using a 10 µL pipet and let samples it for ~1 min more. Do not let it dry completely.
  11. Add 50 µL RNAse-free water and resuspend pellet by pipetting. To resuspend, it helps to aim the water on the “pointy” side of the pellet so it easily comes off the bottom.
  12. Incubate for 15 minutes at 56°C to fully resuspend RNA.

Quantify RNA:

  1. While samples are incubating, prepare for Qubit quantification. Label a Qubit tube for each sample and two standards.
  2. Use the Qubit to calculate the amount of buffer and reagent to use for all samples, two standard, and an extra tube (overage).
  3. Combine the RNA Broad Range buffer and reagent. Add 190 µL of combined reagent mix to each standard tube, and 198 µl of working stock to each sample tube.
  4. Add 10 µL of Standard 1 to the correct Qubit tube. Repeat for Standard 2.
  5. Remove RNA samples from heat block and vortex gently. Add 2 µL of RNA to the correct Qubit tube.
  6. Vortex Qubit tubes and incubate at room temperature for 2 minutes.
  7. Read standards, then samples, with Qubit.

2025-04-18

Notes

  • Was going to start extractions but I didn’t have any time! I did, however, sort all my samples and put aside all of the tissue samples that I need to extract

2025-04-21

Let’s see how many samples I can get done before the end of the month!

Notes

  • Started RNA extractions for 8 samples and a blank
    • 6/29: 25-056, 25-194, 25-049, 25-203, 15-152, 25-167, 25-120, 15-091
    • All sampled used for 25-194 and 25-203
    • 15-152: Miiiight have used a dirty blade end from 25-203 to subsample!

2025-04-22

Notes

  • Finished RNA extractions for 8 samples and a blank
    • 6/29: 25-056, 25-194, 25-049, 25-203, 15-152, 25-167, 25-120, 15-091
    • Made 20 µL of 75% EtOh
    • Qubit: 12 µL dye, 2388 µL buffer
    • Did not vortex samples 15-152, 25-167, and 25-120 before adding to the Qubit working mix
  • Started RNA extractions for 12 samples and a blank
    • 7/5: 15-154, 25-109, 25-180, 25-047, 5-014, 5-066, 5-127, 5-181
    • 8/9: 15-033, 25-046, 25-050
    • 25-180: Top is labelled 25-177, tube is 25-180
    • 25-047: Tissue tube is labelled as 25-048
    • 5-066: Tube is labelled 5-060. Spilled RNALater from tube when removing tissue
    • 25-050: Forgot to blot RNALater before smushing with pestle!
  • Labelled a new box for samples I’ve already processed

Results

Table 1. RNA concentrations (µg/mL). S1 = 435.21, S2 = 7961.68

Sample µg/µL
25-056 0.088
25-194 0.0645
25-049 0.0674
25-203 0.0360
15-152 0.127
25-167 0.0560
25-120 0.195
15-091 0.0749
EB TOO LOW

I have decent looking yields! Carolyn and Dain had some issues with the Qubit this morning, so I followed their advice and ensured all Qubit reagents were at room temperature for 30 minutes before I ran the reactions.

2025-04-23

Notes

  • Finished RNA extractions for 12 samples and a blank
    • 7/5: 15-154, 25-109, 25-180, 25-047, 5-014, 5-066, 5-127, 5-181
    • 8/9: 15-033, 25-046, 25-050
    • 15-033: Disturbed pellet when removing supernatant! Put back in the centrifuge with the EB to spin for 3 minutes to repellet
    • Centrifuge was at 16ºC at the start of the 8 minute spin, but cooled down quickly
    • Made 32 µL of 75% EtOH
    • Qubit: 16 µL dye, 3184 buffer.
  • Started RNA extractions for 16 samples and a blank
    • 6/28: 25-051, 5-133, 15-161
    • 7/24: 25-055
    • 8/9: 25-054, 25-115, 25-195 25-175, 25-052
    • 8/16: 5-003, 5-131, 5-125, 5-075, 5-010, 5-015
    • All sample used for 5-003, 5-131, 5-133
    • 25-195: Tough to pipet homogenize after adding Trizol
    • 5-015: Dropped sample on the counter after blotting!
    • Added 1000 µl of Trizol after smushing sample instead of 2 rounds of 500 µL
  • Labelled a new box for samples I’ve already processed

Results

Table 2. RNA concentrations (µg/mL). S1 = 331.47, S2 = 7993.72

Sample µg/µL
15-154 0.0944
25-109 0.133
25-180 0.0963
25-048 0.0556
25-169 0.104
5-014 0.109
5-066 0.104
5-127 0.0901
5-181 0.0868
15-033 0.0750
25-046 0.0612
25-050 0.0635
EB TOO LOW

Still got a decent yield out of the sample I didn’t blot! So that’s good to know.

2025-04-24

Notes

  • Finished RNA extractions for 25-051, 5-133, 15-161, 25-055, 25-054, 25-115, 25-195 25-175, 25-052, 5-003, 5-131, 5-125, 5-075, 5-010, 5-015
    • Qubit: S1 = 323.52, S2 = 8471.75
  • Started RNA extractions for 20 samples and 1 blank
    • 6/28: 15-092, 5-009, 5-122, 25-0202
    • 6/29: 5-004, 5-121, 5-069
    • 7/24: 25-112, 25-171, 25-177, 25-114, 5-012, 5-130, 5-123, 5-002
    • 8/9: 25-170, 25-116, 5-008
    • 8/16: 5-005, 5-061
    • 25-177: Dropped sample on the counter
    • 5-012: All sample used
    • 3 samples didn’t have a clear phase separation, so I re-spun the samples for 15 more minutes after shaking them up: 25-114, 25-116, 5-008

Results

I’m tired so from now onwards results can be found in this spreadsheet.

Yields were low for 5-125 (0.0206) and 5-075 (0.0364). I think these tissues were the smallest. If needed, I can re-extract based on the prepared libraries.

2025-04-25

Notes

  • Finished RNA extractions for 15-092, 5-009, 5-122, 25-0202, 5-004, 5-121, 5-069, 25-112, 25-171, 25-177, 25-114, 5-012, 5-130, 5-123, 5-002, 25-170, 25-116, 5-008, 5-005, 5-061
    • There was a small black hair sample 25-202 with the precipitate
    • Qubit: 24 µL dye, 4776 µL buffer
      • S1: 318.85, S2: 8122.68
  • Started RNA extractions for 14 samples and 1 blank
    • 6/28: 25-168, 25-110, 25-119, 5-189, 5-182
    • 6/29: 5-063, 5-007
    • 7/24: 5-064, 5-071
    • 8/9: 25-174, 25-060, 25-179
    • 8/16: 5-073, 5-126
    • I forgot to pre-cool the centrifuge, so samples were incubated for 10-15 additional minutes with BCP while I waited for the centrifuge to cool
    • We are out of Trizol!

Results

Concentrations are in this spreadsheet! The samples that I ended up re-spinning had higher concentrations than some of the ones that had weak phase separation that I didn’t re-spinning. I’ll keep this in mind for future extractions.

There are a few samples that had < 0.05 µg/µL so I’ll see how the library preparation goes for those samples.

2025-04-29

Notes

  • Finished RNA extractions for 25-168, 25-110, 25-119, 5-189, 5-182, 5-063, 5-007, 5-064, 5-071, 25-174, 25-060, 25-179, 5-073, 5-126
    • Qubit: 18 µL dye, 3582 µL buffer
      • S1: 337.20, S2: 7977.68
  • Started RNA extractions for 14 samples and 1 blank
    • 6/28: 25-053
    • 6/29: 5-197
    • 7/5: 25-048
    • 7/24: 25-059
    • 8/9: 25-113, 25-186, 25-185, 25-172, 5-067, 5-128, 5-188, 5-001
    • 8/16: 5-135, 5-006
    • Needed to use Ann’s Trizol for all samples since we were out!
    • Placed the aqueous layer of 25-113 in a tube with 25-048’s layer, so I redid these two samples with an extra blank. I now have 16 tubes (14 samples + 2 blanks) total
    • 25-048 all used up

Results

Again, yields are looking good! I used slightly less tissue this time which is why my yields are not as high as they have been for the past few rounds.

2025-04-30

Notes

  • Finished RNA extractions for 25-168, 25-110, 25-119, 5-189, 5-182, 5-063, 5-007, 5-064, 5-071, 25-174, 25-060, 25-179, 5-073, 5-126, and 2 blanks
    • Qubit: µL dye, µL buffer
      • S1: 365.95 , S2: 8148.72

Results

Today I had the lowest yields across all samples! I think this is because when I was pulling tissue, I noticed I was taking less than usual with the intention of having enough tissue left for most samples in case I needed to re-extract them.

BUT AT LEAST I AM DONE.

Going forward

  1. Find all metabolomics samples
  2. Send samples for metabolomics
  3. Examine HOBO data from 2023 experiment
  4. Demographic data analysis for 2023 paper
  5. Start methods and results of 2023 paper
Written on April 21, 2025