Testing Paxgene Fixative, PAXgene Tissue Stabilizer, Sectioning, Decalcification, and Extraction of DNA/RNA using Zymo Quick DNA/RNA Protocol from Stabilized Samples (Time Course), April 13, 2023

Testing Decalcification with PAXgene Fixative and PAXgene Tissue Stabilizer

Over the last few weeks I have been the PAXgene Fixative and PAXgene Tissue Stabilizer for adult coral sample fixation with better-preserved RNA and DNA than traditional formalin/formaldehyde/Z-fix fixation. I now want to attempt to decalcify coral samples after PAXgene fixation.

The plan is to fix more fragments in PAXgene fixative as before, but now to create small disks, or “sections,” of the fixed coral with a razor blade and to decalcify these disks using various methods. Some disks will stay un-decalcified as controls. Then I will attempt to extract RNA and DNA from all the disks and see if decalcification method affected RNA and DNA quality or quantity.

Here are the different trials:

• No decalcifcation (control)
• HCl decalcifcation
• HCl decalcifcation, on ice
• EDTA decalcifcation - in straight 0.5 M EDTA
• EDTA decalcifcation, on ice
• Whole branchlet decalcified in HCl
• Whole branchlet decalcified in EDTA - in straight 0.5 M EDTA

Order of operations:

1. Fix branchlets in PAXgene fixative for 24 hours.
   1. Fixation could probably be cut to overnight?
2. Transfer branchlets to PAXgene stabilizer for at least 2 hours at 4ºC to allow for penetration of stabilizer into the tissues. I did 24 hours
3. Cut branchlets into disks and record weight - divide into various decalcification treatments.
   1. TWO per treatment, one for RNA and one for histology
4. After decalcification, samples should go straight into labelled tubes with PAXgene stabilizer, possibly with another 4 ºC incubation step to allow for PAXgene stabilizer penetration into tissue, and then stored at -80ºC. (We can also test putting them in the fridge)
   1. Decalc is done when disk floats and there is no CaCO3 left visually
5. Then extract these using Zymo RNA extraction kit. Weighing starting material to try to keep it consistent is a good idea.
   1. Bead-beat 2 mins with DNA/RNA shield or not?

What I did (4/13/23):

I fixed 5 branchlets from the same coral from my last test in the exact same protocol followed described there.

After 24 hours in PAXgene tissue stabilizer at 4ºC, I cut 12 fragments of 0.2 - 0.3 g each in PAXgene stabilizer (using clippers was way easier than razor blade). 2 fragments went into each tube (one for histology and one for RNA):

• No decalcifcation (control)
• No decalcifcation (control), on ice
• HCl decalcifcation (10%)
• HCl decalcifcation (10%), on ice
• EDTA decalcifcation - in straight 0.5 M EDTA
• EDTA decalcifcation, on ice

I also decalcified a whole brachlet in HCl (5mL) and one whole brachlet in EDTA (5mL).

Started decalcification at 13:30 on 4/13/23. A few bubbles in the HCl treatments but not in the control or EDTA. After 1.5 hour, all of the samples on ice had barely any bubbles so I decided the best course of treatment was to move these to 4ºC in the cold room so they could stay as cold as possible while slowly decalcifying. As of 4 hours in, they still have a lot of skeleton so I am leaving them overnight.

As of 7:30 AM the next morning (4/14/23), they were still not fully decalcified.

Refreshed solutions after 24 hours (13:30 on 4/14/23) and moved all samples to larger 5mL tubes with fresh solutions to maximize contact between the solution and the samples. (The non-full branchlet samples were originally in 1.5 mL tubes)

On Saturday 4/15/23 at 11:30 AM the fragments were 90% decalcified and I removed all fragments from their decalcification treatments (or control) and moved each fragment into an individual 2mL screw top tube with 1mL of PAXgene stabilizer (all with sterilized forceps).

There appeared to be a reaction between the EDTA samples and the PAXgene stabilizer (precipitation) so I moved the tissue into new tubes of stabilizer to minimize any extra excess EDTA in the mixture.

Decalcified whole branch from EDTA:

Decalcified whole branch from HCL (probably could’ve gone a little longer but wanted to keep the time for all samples together):

All tubes (14) placed into -80 ºC freezer until extraction / histology prep.

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RNA Extraction Test

6 samples:

• No decalcifcation (control)
• No decalcifcation (control), on ice
• HCl decalcifcation
• HCl decalcifcation, on ice
• EDTA decalcifcation - in straight 0.5 M EDTA
• EDTA decalcifcation, on ice

Qubit Results

• Used Broad range dsDNA and RNA Qubit Protocol
• All samples read twice, standard only read once

Concentrated RNA from 100 uL to 25 uL using Zymo clean and concentrate RNA kit and re-qubit on 4/25/23, results are below. (Very similar to original Qubit, but now control + cold room has RNA concentration of 14.1 ng/uL, was 0 like the others before.)

colony_id	DNA_QBIT_AVG	RNA_QBIT_AVG
control	0	0
control + cold room	0	14.1
EDTA	0	0
EDTA + cold room	0	0
HCl	0	0
HCl + cold room	0	0

DNA and RNA Quality Check: Gel, using Kristina’s gel protocol at the bottom of this page.

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Questions:

Do we want to buy tissue casettes? The PAXgene containers can hold 4 casettes.

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Example protocols:

• HCl decalcifcation: Jill and Chloe protocol
  • Corals fixed in Zfix (10% aqueous buffered zinc formalin) with filtered seawater (FSW), 1:4 ratio of Z-fix:FSW
  • Z-fix replaced with 70% ethanol
  • Decalcified in 10% HCl
  • Then stored in 20% Z-fix
• EDTA decalcifcation: Katie Barrot protocol, based on this
  • Corals fixed in S22 buffer with 3% paraformaldehyde (PFA) overnight at 4 °C
  • “Decalcify by transferring fragment to Ca-free S22 buffer with 0.5 M EDTA and 0.5% paraformaldehyde and incubate at 4 °C. Replace the buffer daily until the skeleton is completely dissolved (~7 days depending on size of fragment and thickness/density of skeleton).”
    • Ca-free S22 buffer + 0.5 M EDTA
      • 450 mM NaCl
      • 10 mM KCl
      • 58 mM MgCl2
      • 0.5 M EDTA
      • 100 mM Hepes (pH 7.8)
        • Note: Adjust pH of Hepes buffer with NaOH.
  • Then dehydrated in ethanol series starting with 50% ethanol
• EDTA decalcification: Liew et al laser microdissection protocol, based on this basically the exact same as above
  • Fixed in 3% paraformaldehyde in S22 buffer at 4°C overnight
  • Decalcified using 0.5 M EDTA in Ca-free S22 at 4°C.
  • They were then dehydrated in an ethanol series and embedded in Paraplast.
• EDTA decalcification: Liu et al tissue clearing, similar to both EDTA protocols above, but concentrations slightly different
  • Fixed in 4% paraformaldehyde in filtered sea water (FSW)
  • Washed with 0.01‐M PBS 3x at room temperature
  • Decalcification in 10% EDTA at pH 7.4.
    • “The solution was replaced twice every day until the whole skeleton was removed.”
  • “Methanol was used for decolorising and permeabilisation. The samples were washed with PBS thrice and dehydrated in 50%, 75%, 90%, and 100% methanol in 0.01‐M PBS”
• Morse’s solution decalcifcation
  • Nikki Traylor-Knowles 2017 Paper Protocol
  • Coral fixed in in 4% paraformaldehyde in filtered seawater
  • Washed in PBS
  • Stored in methanol at −20°C
  • Samples prepared by a histology lab (IDEXX)
  • “Samples were decalcified using Morse’s solution (25% formic acid, 10% sodium citrate)”
  • Then dehydrated and paraffin embedded