2026-03-10 Qiagen RNeasy Extraction Tests, Cryosectioned tissue optimization

Extracting RNA from PAXgene fixed tissue at various stages of decalcification

Timeline

  • 3/10/26: Corals fixed
  • 3/11/26: Fixative replaced with stabilizer
  • 3/12 - 3/14: 48 hours of decalc, MON and 3/4 POC embedded
  • 3/12 - 3/15: 72 hours of decalc for 2 POR + 1 POC
  • 3/12 - 3/16: 96 hours of decalc for 3 POR
  • 3/17 & 3/19: Sectioning
  • 3/20: All slides H&E Stained
  • 3/20 + 3/25: Imaging with EVOS7000
  • 3/25: Extraction of 6 scrolls

Protocol: Qiagen RNeasy Kit

Continued testing from first test of this kit for RNA QC here and 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 (tissue scroll extraction protocol)
    1. Tubes were taken out of freezer on dry ice and 150 uL RLT (w/ 1% BME - 1000 + 10 uL) was added and the scrolls were dissolved and mixed with a wide-bore tip
    2. Then, 290 μl RNase-free water and 10 μl Proteinase K were added and briefly mixed by vortex
    3. Incubate the tissue on a shaker–incubator for 15 min at 56°C and 1400 rpm
    4. Centrifuge for 3 min at maximum speed (but do not exceed 20,000 x g).
  4. Carefully remove 350 uL of the supernatant by pipetting, and transfer it to a new 1.5 mL tube
  5. Add 1 volume of 70% ethanol* to the cleared lysate, and mix immediately by pipetting.
  6. 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.
  7. 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.
  8. Add 500 µl Buffer RPE to the RNeasy spin column.
  9. Close the lid gently, and centrifuge for 30 s at 15,000 rcf to wash the spin column membrane. Discard the flow-through.
  10. 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.
  11. 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.
  12. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied)
  13. 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.
  14. If higher concentration is desired, re-apply this eluate to the filter membrane and repeat the centrifuge step.

3/16/26: All final blocks embedded, details in google sheet and notebook

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

Embedded blocks were sectioned 3/17 and 3/19:

Block_Label Embedding notes - how to section Block_Description Section_Date Section_Notes Block_Status Slide_1_label Slide_1_Status Slide_2_label Slide_2_Status Slide_3_label Slide_3_Status        
POC_1-A discs 48 hr OCT:30% test 20260317 Second slide is better - looking pretty good in -80 0317_02 H&E Stained 3/20 0317_03 H&E Stained 3/20            
POC_2-A discs 48 hr 20260317 Tore a lot, one branch I think in -80 0317_04 H&E Stained 3/20 0317_05 H&E Stained 3/20            
POC_3 one disc 72hr, no sucrose 20260317 Sectioned super well in -80 0319_10 H&E Stained 3/20 0319_11 H&E Stained 3/20 0319_12 H&E Stained 3/20        
POC_4 one disc 72 hr OCT:30% test 20260317 Sectioned super well; collected 3 tubes, use #3 in -80 0317_08 H&E Stained 3/20 0317_09 H&E Stained 3/20 0319_09 H&E Stained 3/20        
Block_Label Embedding notes - how to section Block_Description Section_Date Section_Notes Block_Status Slide_1_label Slide_1_Status Slide_2_label Slide_2_Status Slide_3_label Slide_3_Status
POR_1 section 90 degrees 72hr, no sucrose 20260319 went ok; got automatic sectioning to work in -80 0319_08 H&E Stained 3/20        
POR_2 section 90 degrees 72 hr OCT:30% test 20260319 tore a little but pretty good in -80 0319_06 in -80 0319_07 H&E Stained 3/20    
POR_3 section 90 degrees 96 hr OCT:30% test 20260319 2nd slide has one section at 20 um in -80 0319_03 H&E Stained 3/20 0319_04 H&E Stained 3/20    
POR_4 section 90 degrees 96 hr OCT:30% test, expect rip 20260319 took about an hour to get in the groove, but when it did it worked great in -80 0319_01 H&E Stained 3/20 0319_02 H&E Stained 3/20    
POR_5 section 90 degrees 96 hr 20260319 tore a lot, tissue coming out of OCT in -80 0319_05 in -80        
Block_Label Embedding notes - how to section Block_Description Section_Date Section_Notes Block_Status Slide_1_label Slide_1_Status Slide_2_label Slide_2_Status Slide_3_label Slide_3_Status          
MON_1 section like normal 48 hr OCT:30% test - had some skeleton, expect rippage 20260319 2nd tube is better in -80 0319_10 H&E Stained 3/20 0319_11 H&E Stained 3/20 0319_12 H&E Stained 3/20          
MON_2 section 90 degrees 48 hr OCT:30% test 20260317;20260319 Hard to section :/ Ripped a lot. sectioned second slide at 20 um. Tried again 3/19. coming out of OCT a lot in -80 0317_08 H&E Stained 3/20 0317_09 H&E Stained 3/20 0319_09 H&E Stained 3/20          
MON_3 section 90 degrees 48 hr 20260317 Hard to section :/ Ripped a lot because it was so far to the bottom of the mold. sectioned at 20 um in -80 0317_10 H&E Stained 3/20                  
MON_4 section 90 degrees 48 hr, expected to be messed up though 20260319 Coming out of OCT too much. 1 tube but no slides in -80               In -80 In -80 H&E Stained 3/2 H&E Stained 3/2

3/25/26: Tissue scrolls extraction

2026-03-25

sample_id concentration RIN
Scroll - POC_3 (EDTA 72hr, no sucrose) 2.47 ng/uL 5.8
Scroll - POC_4 (EDTA 72hr, Sucrose w/ extra 30% Sucrose:OCT incubation) 3.24 ng/uL 5.2
Scroll - POR_2 (EDTA 72hr, Sucrose w/ extra 30% Sucrose:OCT incubation) 1.13 ng/uL 6.8
Scroll - POR_3 (EDTA 96hr, Sucrose w/ extra 30% Sucrose:OCT incubation) 2.18 ng/uL 7.8
Scroll - MON_2 (EDTA 48hr, Sucrose w/ extra 30% Sucrose:OCT incubation) 8.22 ng/uL 4.9
Scroll - MON_3 (EDTA 48hr, Sucrose w/o extra 30% Sucrose:OCT incubation) 7.77 ng/uL 4.8

Full results can be found here

sample_id concentration RIN example pic slidescan morphology assessment/notes
Scroll - POC_3 (EDTA 72hr, no sucrose) 2.47 ng/uL 5.8 POC_3_2_40x_polyp_tile POC_3_2_4x_slideScan_small calcioblast morphplogy not preserved as well as POC_4
Scroll - POC_4 (EDTA 72hr, Sucrose w/ extra 30% Sucrose:OCT incubation) 3.24 ng/uL 5.2 POC_4_2_20x_nice_ring POC_4_2_4x_slideScan looks really really great
Scroll - POR_2 (EDTA 72hr, Sucrose w/ extra 30% Sucrose:OCT incubation) 1.13 ng/uL 6.8 POR_2_2_20X_Section2_4x4bin POR_2_2_4X_SlideScan_all_Sep looks great
Scroll - POR_3 (EDTA 96hr, Sucrose w/ extra 30% Sucrose:OCT incubation) 2.18 ng/uL 7.8 POR_3_2_20x_Section6 POR_3_2_4x_SlideScan looks really really great
Scroll - MON_2 (EDTA 48hr, Sucrose w/ extra 30% Sucrose:OCT incubation) 8.22 ng/uL 4.9 MON_2_1_10x_Section4 MON_2_1_4x_slideScan really jumbled tissue, hard to tell what is what
Scroll - MON_3 (EDTA 48hr, Sucrose w/o extra 30% Sucrose:OCT incubation) 7.77 ng/uL 4.8 MON_3_1_10x_section2 MON_3_1_4x still not stellar, but maybe the best MON sections we’ve ever gotten
Written on March 10, 2026