Testing Paxgene Fixative, PAXgene Tissue Stabilizer, and Extraction of DNA/RNA using Zymo Quick DNA/RNA Protocol from Stabilized Samples (Time Course), March 21, 2023

Testing PAXgene Fixative and PAXgene Tissue Stabilizer

In this protocol, I tested the use of PAXgene Tissue Fix Containers for coral. I used a sample of Pocillopora acuta from our Putnam Lab Wetlab.

On March 20, 2023, I cut off four branchlets of about 5 mm long and carried these to the lab in a 5mL tube filled with seawater from the aquarium. In the fume hood, I transferred (with sterile forceps) all four branchlets to a labelled PAXgene Tissue Fix container.

I fixed these four branchlets for 24 hours at room temperature (in the fume hood). The color from the fragments immediately begain leaching into the solution and after 2 hours the fragments looked completely white.

After 24 hours, I poured off the PAXgene fixative into a labelled waste container (in fume hood) and immediately filled the container with PAXgene stabilizer (350mL Ethanol added to concentrate and mixed before starting). Then the container was transferred to the 4 ºC flammable fridge. I will remove one sample in 2 hours for DNA/RNA extraction, one in one week, and one in two weeks. One sample was transferred into a 2mL screw-cap vial filled with PAXgene tissue stabilizer and placed at -80 ºC after the 2 hour incubation at 4 ºC to test this storage method.

Extraction of DNA/RNA using Zymo Quick DNA/RNA Protocol from Stabilized Samples (Time Course), March 21, 2023

DNA/RNA Extraction Protocol Link

Followed this protocol exactly, but followed steps for samples preserved in RNAlater. Bead-beat for 2 minutes.

Extraction, Day 1:

2 hours after pouring off the PAXgene fixative and replacing with PAXgene stabilizer (samples maintained at 4 ºC during this time), I removed one fragment from the PAXgene stabilizer and immediately transferred it to a 2 mL tube filled with 1 mL of DNA/RNA shield and beads and continued as described in the protocol for processing samples in RNAlater.

Qubit information for 3/21/23: DNA Standards: 210.45 (S1) & 24497.80 (S2)

RNA Standards: 414.90 (S1) & 8937.18 (S2)

Extraction, Week 1 + -80 ºC:

At 12pm on 3/28/23, I started the extraction of one branchlet that had been kept in the PAXgene stabilizer at 4 ºC for one week exactly and one that had been kept at -80 ºC for one week exactly. Same protocol as prior.

Qubit information for 3/28/23: DNA Standards: 193.30 (S1) & 20909.36 (S2)

RNA Standards: 399.23 (S1) & 8736.93 (S2)

Extraction, Week 2

At 12pm on 4/6/34, I started the extraction of one branchlet that had been kept in the PAXgene stabilizer at 4 ºC for two weeks. It was the largest branchlet of the 4 with the 3 nubbins.

Qubit information for 4/6/23: DNA Standards: 197.48 (S1) & 22870.14 (S2)

RNA Standards: 406.98 (S1) & 8665.72 (S2)

Qubit Results

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

colony_id	DNA_QBIT_1	DNA_QBIT_2	DNA_QBIT_AVG
Day 1	20.6	20.6	20.6
Week 1	6.46	6.50	6.48
Week 2	3.46	3.44	3.45
-80C	20.2	20.6	20.4

---

colony_id	RNA_QBIT_1	RNA_QBIT_2	RNA_QBIT_AVG
Day 1	31.2	31.2	31.2
Week 1	12.0	12.4	12.2
Week 2	14.0	14.6	14.3
-80C	27.0	27.2	27.1

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

Gel from 3/21/2023, of DNA and RNA extracted from the “Day 1” Sample

Gel from 3/28/2023, of DNA and RNA extracted from the “Week 1” Sample and “-80 ºC” Sample, in that order.

Interesting that the -80 bands are brighter than the Week 1 @ 4 ºC in both the RNA and DNA, but the DNA looks smearier than the Week 1 band… This could be a quantity smear not a quality smear.

Gel from 4/6/2023, of DNA and RNA extracted from the “Week 2” Sample

Notes

Okay, so it seems like keeping these in the -80 and extracting as quickly as possible is probably a good idea.

I want to try to figure out a decalcification protocol for these samples. One paper I read did this post-parrafin embedding, but in my experience most coral fixation protocols do it before.

3/29/23: Called Qiagen was pushed up to R & D team, waiting on email from them.

Questions for Lab Meeting

We normally dilute fixative with seawater or prepare PFA in seawater buffer - do you think we should be doing so with these solutions?

Feedback from Qiagen R & D:

“We have tried to decalcify bone samples after fixation with PAXgene Tissue. Using HCl (1M) for decalcification after tissue fixation is in principle possible, but morphology as well as RNA quality is compromised. An alternative to acid would be incubation in EDTA, but this takes longer and also did not lead to optimal results. DNA seems least affected.”