2026-02-19 Qiagen RNeasy Extraction Tests, PAXgene fixed tissue at various stages of decalcification

Extracting RNA from PAXgene fixed tissue at various stages of decalcification

Timeline

timeline

Protocol: Qiagen RNeasy Kit

Continued testing from first test of this kit for RNA QC here

Notes:

  • β-Mercaptoethanol (β-ME) must be added to Buffer RLT before use. Add 10 µl β-ME per 1 ml Buffer RLT. Dispense in a fume hood and wear appropriate protective clothing.
  • Buffer RPE is supplied as a concentrate. Before using for the first time, add 4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a working solution.

Procedure:

  1. Make sure all buffers are prepared appropriately
  2. Label tubes + clean bench
  3. Tissue prep steps
    1. For PAXgene-fixed tissues in PAXgene stabilizer:
      1. Place tissue in 500 µl Buffer RLT with BME added (500 + 5 uL) with 5 mm of beads
      2. Bead beat for 2 mins
      3. For non-Pro-K digested samples (testing 1/2 and 1/2)
        1. Spin down briefly on mini centrifuge and proceed to step 4
      4. For Pro-K digested samples (testing 1/2 and 1/2)
        1. Add 960 µl RNase-free water to cell lysate suspension. Then add 40 µl Proteinase K and mix by vortexing for 5 s.
        2. Incubate at 45 ºC for 15 min at 1400 rpm
      5. Proceed to step 4
  4. Transfer 400 uL to a new 1.5 mL tube and Centrifuge the lysate for 3 min at full speed.
  5. Carefully remove 350 uL of the supernatant by pipetting, and transfer it to a new 1.5 mL tube
    1. Remove as much as you can without disturbing pellet, use same volume of ethanol in next step
  6. Add 1 volume of 70% ethanol* to the cleared lysate, and mix immediately by pipetting.
  7. Transfer up to 700 µl of the sample to an RNeasy spin column placed in a 2 ml collection tube (supplied).
    1. Close the lid gently, and centrifuge for 30 s at 15,000 rcf. Discard the flow-through.
    2. If the sample volume exceeds 700 µl, centrifuge successive aliquots in the same RNeasy spin column. Discard the flow-through after each centrifugation.
  8. Add 700 µl Buffer RW1 to the RNeasy spin column.
    1. Close the lid gently, and centrifuge for 30 s at 15,000 rcf to wash the spin column membrane. Discard the flow-through.
  9. Add 500 µl Buffer RPE to the RNeasy spin column.
  10. Close the lid gently, and centrifuge for 30 s at 15,000 rcf to wash the spin column membrane. Discard the flow-through.
  11. Add 500 µl Buffer RPE to the RNeasy spin column.
    1. Close the lid gently, and centrifuge for 1 min at 15,000 rcf to wash + dry the spin column membrane.
  12. Place the RNeasy spin column in a new 2 ml collection tube (supplied), and discard the old collection tube with the flow-through.
    1. Close the lid gently, and centrifuge at full speed for 2 min.
  13. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied)
  14. Add 50 µl RNAse-free water directly to the spin column membrane. Close the lid gently, and centrifuge for 1 min at 12,000 rcf to elute the RNA.
  15. If higher concentration is desired, re-apply this eluate to the filter membrane and repeat the centrifuge step.

2/19/26: Pre-decalc samples, testing ProK digestion vs. no digestion

Exact protocol written above.

2026-02-19

sample_id concentration RIN
POC_1 96.9 ng/uL 9.1
POC_1_ProK 91.6 ng/uL 8.4
POR_1 44.9 ng/uL 8.5
POR_1_ProK 40.8 ng/uL 8.3
MON_1 144 ng/uL 8.5
MON_1_ProK 168 ng/uL 5.8

Full results can be found here

2/21/26: First 24 and 48 hours of decalcification results

  • Tissue sampled throughout experiment was collected in 500 uL RLT (w/ 1% BME) in a 2mL tube with beads and bead-beat for 2 mins before being frozen at -80ºC
  • Tubes were thawed at room temperature and bead-beat ~1 minute to ensure homogenous thawing and to clear any precipitate
  • Same protocol followed as above, but without the proK digestion
  • Notes
    • For POR and MON, these tubes had a lot of tissue and were pretty cloudy/dark in the lysate.
    • Formed big pellets during the 3 min spin suggesting that maybe there was too much tissue input
    • During elution, the POR columns clogged, potential mucus carry-over.
      • Added another 50 uL on top instead of re-eluting with same 50uL, final volume was closer to 75 uL
    • This is not great news. Even with just 24 hours of decalc, when none of the corals are decalcified, we get substantial decrease in RIN below what we are aiming for.

2026-02-21

sample_id concentration RIN
POC_2 (EDTA 6hr) 148 ng/uL 7.1
POC_3 (EDTA 24hr) 160 ng/uL 7.4
POC_4 (EDTA 24hr+2 hrs sucrose) 88.9 ng/uL 4.6
POC_5 (EDTA 48hr) 82.4 ng/uL 7.5
MON_2 (EDTA 24hr) 155 ng/uL 6.0
MON_3 (EDTA 48hr) 194 ng/uL 5.4
POR_2 (EDTA 24hr) 52.4 ng/uL 6.5
POR_3 (EDTA 48hr) 35.6 ng/uL 6.2

Full results can be found here

2/22/26: First 24 and 48 hours of decalcification results, re-extraction of POR + MON

  • Because of the big pellets formed, I thought maybe the filter clogging could be fixed by re-extracting from those tubes with more RLT added to dilute the lysate. Re-extracting POR_2, POR_3, MON_2, and MON_3
    • Added another 500 uL RLT (w/ 1% BME) to the tubes as they thawed + pipetted to mix
    • Bead-beat ~1 minute to ensure homogenous thawing and to clear any precipitate
    • Moved 450 uL to a new tube and centrifuged for 3 mins to pellet debris, then repeated this with 400 of the supernatant from that spin to try to minimize any debris carry-over into columns.
    • Same protocol followed as above, but without the proK digestion
  • As seen below, RINs improved slightly but not substantially, and large gDNA-appearing peaks still occur. I might try one more time with a gentle ProK digestion
    • This is not great news. Even with just 24 hours of decalc, when none of the corals are decalcified, we get substantial decrease in RIN below what we are aiming for.

2026-02-22

sample_id concentration RIN
POR_2 (EDTA 24hr) 70.7 ng/uL 6.2
POR_3 (EDTA 48hr) 89.3 ng/uL 6.3
MON_2 (EDTA 24hr) 137 ng/uL 6.1
MON_3 (EDTA 48hr) 142 ng/uL 5.6

Full results can be found here

Total count of extractions used from this kit (50 preps):

  • 6+6+8+4 = 24
  • If I re-extract the 4 POR+MON again with Proteinase K and then extract the final 8 tubes, I will have used 36/50 preps from the kit without yet using any of the preps for QC-ing tissue sections. Need to think strategically about which samples to do.

3/2/26: 72 hours decalcification extraction

  • Tissue sampled throughout experiment was collected in 500 uL RLT (w/ 1% BME) in a 2mL tube with beads and bead-beat for 2 mins before being frozen at -80ºC
  • Tubes were thawed at room temperature and bead-beat ~1 minute to ensure homogenous thawing and to clear any precipitate
  • Same protocol followed as above, but without the proK digestion

2026-02-21

sample_id concentration RIN
POC_6 (EDTA 72hr, no sucrose) 131 ng/uL 7.3
POC_7 (EDTA 72hr, Sucrose 3hr) 101 ng/uL 5.0
MON_4 (EDTA 72hr, no sucrose) 212 ng/uL 5.4
MON_5 (EDTA 72hr, Sucrose 3hr) 143 ng/uL 5.0
POR_4 (EDTA 72hr, no sucrose) 71.1 ng/uL 7.1
POR_5 (EDTA 72hr, Sucrose 3hr) 65.9 ng/uL 7.5

Full results can be found here

2/22/26: All final blocks embedded, details in google sheet and notebook

  • This was more rushed than I’d like due to the blizzard on 2/23, several of the POR and MON were not fully decalcified. But it will still give us RNA quality info.

https://docs.google.com/spreadsheets/d/1y8tZUTPmXJmq1QOfvcBU3mhVc6VYH7Xe-fA4zXSnTZ4/edit?usp=sharing

Embedded blocks were sectioned 2/26/26 and 2/27/26:

Block_Label Block_Description Section_Date Section_Notes Block_Status Slide_1_Status Slide_2_Status Slide_3_Status
POC_4 EDTA-24hr-Suc120 2/26/26 Was surpisingly good In -80 H&E Stained 2/26 H&E Stained 2/26 N/A
POC_5 EDTA-48hr-Suc-210 2/26/26   In -80 H&E Stained 2/26 H&E Stained 2/26 N/A
POC_6 EDTA-72hr-noSuc 2/26/26 Was surpisingly good In -80 H&E Stained 3/2 H&E Stained 3/2 In -80
POC_7 EDTA-72hr-Suc150 2/26/26   In -80 H&E Stained 3/2 N/A N/A
Block_Label Block_Description Section_Date Section_Notes Block_Status Slide_1_Status Slide_2_Status Slide_3_Status
MON_3 EDTA-48hr-Suc180 2/27/26 Too much skeleton, really hard to section In -80 H&E Stained 3/2 N/A N/A
MON_4 EDTA-72hr-noSuc 2/27/26 Of all the MON, maybe the best? In -80 H&E Stained 3/2 N/A N/A
MON_5 EDTA-72hr-Suc180 2/26/26 Too much skeleton, really hard to section In -80 Threw away, was really bad N/A N/A
MON_6 EDTA-72hr-Suc300 2/26/26 Too much skeleton, really hard to section In -80 H&E Stained 3/2 N/A N/A
Block_Label Block_Description Section_Date Section_Notes Block_Status Slide_1_Status Slide_2_Status Slide_3_Status
POR_3 EDTA-48hr-Suc150 2/27/26 Some skeleton tearing In -80 H&E Stained 3/2 In -80 N/A
POR_4 EDTA-72hr-noSuc 2/27/26   In -80 In -80 H&E Stained 3/2 N/A
POR_5 EDTA-72hr-Suc180 2/27/26 Tore a lot, but I think it was because I put too much tissue in the block In -80 H&E Stained 3/2 N/A N/A
POR_6 EDTA-72hr-Suc300 2/27/26   In -80 In -80 H&E Stained 3/2 H&E Stained 3/2

3/3/26: Tissue scrolls extraction

  • 3-5 Tissue scrolls (30 um thick) were collected from the OCT-embedded blocks of the corals described above that went through various combinations of decalcification + sucrose times. The ones that produced the most viable tissue/section quality were extracted below, but I also have scrolls in the freezer from blocks that sectioned pooly due to incomplete decalcification.
  • Scrolls were collected in 1.5 mL tubes cooled to cryostat temperature and immediately placed on dry ice before transport to -80 ºC
  • Extraction protocol was modified from the standard RNeasy protocol for paxgene-fixed tissue sections in OCT
    • Modifications based on the protocol of the Qiagen PAXgene Tissue RNA/miRNA Kit (handbook and cryo-embedding specific modifications)
    • Tubes were taken out of freezer on ice and 150 uL RLT (w/ 1% BME) was added and the scrolls were dissolved and mixed with a wide-bore tip
    • Then, 290 μl RNase-free water and 10 μl Proteinase K were added and briefly mixed by vortex
    • Incubate the tissue on a shaker–incubator for 15 min at 56°C and 1400 rpm
    • Centrifuge for 3 min at maximum speed (but do not exceed 20,000 x g).
    • Carefully transfer the supernatant to a new 1.5 ml microcentrifuge tube without disturbing the pellet.
    • Add equal volume of 70% ethanol and continue with RNeasy protocol as written.

2026-03-03

sample_id concentration RIN
Scroll - POC_6 (EDTA 72hr, no sucrose) 16.2 ng/uL 6.9
Scroll - POC_7 (EDTA 72hr, Sucrose) 16.4 ng/uL 4.5
Scroll - MON_4 (EDTA 72hr, no sucrose) 40.2 ng/uL 4.8
Scroll - MON_6 (EDTA 72hr, Sucrose 5hr) 38.8 ng/uL 4.1
Scroll - POR_4 (EDTA 72hr, no sucrose) 11.8 ng/uL 7.7
Scroll - POR_5 (EDTA 72hr, Sucrose 3hr) 15.1 ng/uL 7.5
Scroll - POR_6 (EDTA 72hr, Sucrose 5hr) 9.16 ng/uL conc. too low, rerun with HS

Full results can be found here

YAY! We have tissue scrolls with RINs at 6.9 for POC and > 7 for POR. The non-sucrose slides still don’t look like the morphology we want, but this is major progress.

Written on February 19, 2026