ZYMO Quick-DNA/RNA Miniprep Plus Kit Troubleshooting

Goal

• To determine if both DNA and RNA extracts can be obtained from an adult coral homogenate (see airbrushing protocol) and a biopsy of adult tissue and skeleton. The coral used for this test was Porites astreoides collected from Bermuda.

Experimental design

Table 1: Experimental Design to test DNA/RNA extraction methods on 2 types of coral tissues.

Sample #	Sample Type	Coral ID/Vial#	Volume/Size of Sample
1	Homogenate	951	500 mL
2	Homogenate	954	500 mL
3	Fragment	R20-A	~10 mm diameter
4	Fragment	R20-A	~10 mm diameter

Overall workflow

1. Prepare samples
2. Extract and prep DNA with column (elute RNA and Protein)
3. Extract and prep RNA with column (elute Protein)
4. Quantify DNA, RNA, and protein extracts

Protocol preparation

• Using slightly modified Zymo Duet DNA/RNA extraction protocolwhich will extract both DNA and RNA at the same time (Below are summary steps)

Reagents and supplies

• RNase-free Water
• 100% ethanol, ACS grade or better
• 10mM Tris HCl pH 8.0 made with RNase-free water
• ZR BBashing lysis tubes with 0.1mm and 0.5mm beads

Equipment

• Rocking oven that can be set to 55°C
• RNase away and a designated RNase free space
• Tabletop and larger centrifuges for 1.5mL and 50mL tubes capable of 12,000 x g

Clipper Sterilization

• Rub down clippers with:
  1. 10% Bleach solution
  2. DI water
  3. 90% ethanol
  4. RNAse free water

Notes before starting

• Wipe down benchtop with RNase away and have the spray bottle and kimwipes on-hand to use frequently

Sample preparation

Homogenate preparation

• Immediately after taking sample out of the -80°C freezer, add 500μL of DNA/RNA shield
• Add 100 μL of Protenase K digestion buffer
  • Derived from volume of homogenate and 300/30 ratio from Zymo Protocol
• Add 50 μL of Protenase K
  • Derived from volume of homogenate and 300/15 ratio from Zymo Protocol
• Incubate on Thermomixer at 56°C at 600 rpm for 30 minutes
• Remove all liquid (1150 μL) and transfer in 5 mL tubes

Fragment preparation

• Sterilize clippers (as outlined above)
• Clip off ~10mm in diameter of tissue and skeleton and place into ZR BBashing lysis tube
• Add 1000 μL of DNA/RNA shield (may be too much)
• Vortex for ~1 minute
• Remove 500 μL of supernatant and transfer into 5mL tube
  • Large mucus chunks made this step difficult –> potential source of error

DNA Extraction

1. Add equal volumes of DNA lysis buffer as sample volume to the 5 mL tube with sample
   • 1150 μL for the homogenate samples
   • 500 μL for the fragment samples
   • Vortex to mix
2. Transfer 700 μL into DNA filter column (yellow)
   • Centrifuge at 16,000 rcf for 30 seconds
   • Remove flow through liquid and transfer into a new 5 mL tube labeled for RNA
3. Repeat steps 2 until all liquid is gone
4. Add 400 μL of DNA/RNA prep buffer to column
   • Centrifuge at 16,000 rcf for 30 seconds
   • Remove the flow through and transfer to waste
5. Add 700 μL of DNA/RNA wash buffer
   • Centrifuge at 16,000 rcf for 30 seconds
   • Remove the flow through and transfer to waste
6. Add 400 μL of DNA/RNA wash buffer
   • Centrifuge at 16,000 rcf for 2 minutes
   • Remove the flow through and transfer to waste
7. Removed column and place into a new sterile 1.5 mL tube
8. Add 50 μL of 10 mM Tris HCL (warmed to 55°C) directly to filter
   • Incubate at room temperature for 5 minutes
   • Centrifuge at 16,000 rcf for 30 seconds
   • Keep flow through in tube
9. Add another 50 μL of 10 mM Tris HCL (warmed to 55°C) directly to filter
   • Incubate at room temperature for 5 minutes
   • Centrifuge at 16,000 rcf for 30 seconds
   • Discard column and keep flow through in tube
10. Aliquot into appropriate tubes for storage at -20°C

RNA Extraction

1. Add equal volumes of 100% Ethanol as sample volume to the 5 mL tube with sample
   • 1150 μL for the homogenate samples
   • 500 μL for the fragment samples
   • Vortex to mix
2. Transfer 700 μL into RNA filter column (green)
   • Centrifuge at 16,000 rcf for 30 seconds
   • Remove flow through liquid and transfer into a new 5 mL tube labeled for Protein
3. Repeat steps 12 until all liquid is gone
4. Add 400 μL of DNA/RNA wash buffer to column
   • Centrifuge at 16,000 rcf for 30 seconds
   • Remove the flow through and transfer to waste
5. Make DNase I master mix: [75μL DNA digestion buffer and 5μL DNase] X n (sample #)
6. Add 80μL of DNase I master mix directly to the filter of each column. Incubate at room temperature for ~15 minutes
7. Add 400 μL of DNA/RNA prep buffer to column
   • Centrifuge at 16,000 rcf for 30 seconds
   • Remove the flow through and transfer to waste
8. Add 700 μL of DNA/RNA wash buffer
   • Centrifuge at 16,000 rcf for 30 seconds
   • Remove the flow through and transfer to waste
9. Add 400 μL of DNA/RNA wash buffer
   • Centrifuge at 16,000 rcf for 2 minutes
   • Remove the flow through and transfer to waste
10. Removed column and place into a new sterile 1.5 mL tube
11. Add 50 μL of RNAase-free water (warmed to 55°C) directly to filter
    • Incubate at room temperature for 5 minutes
    • Centrifuge at 16,000 rcf for 30 seconds
    • Keep flow through in tube
12. Add another 50 μL of RNAase-free water (warmed to 55°C) directly to filter
    • Incubate at room temperature for 5 minutes
    • Centrifuge at 16,000 rcf for 30 seconds
    • Discard column and keep flow through in tube
13. Aliquot into appropriate tubes for storage at -80°C

Quantification

Qubit

• For the Qubit readings, 10 μL of each standard were used and 1 μL of each sample were used.

Table 2: Qubit readings for the extracted DNA and RNA samples.

	Standard 1	Standard 2	#1 (ng/uL)	#2 (ng/uL)	#3 (ng/uL)	#4 (ng/uL)
DNA	205.05	22675.65	16.70	13.00	8.24	3.03
DNA	NA	NA	16.10	13.00	8.10	2.98
RNA	393.14	11692.94	10.20	11.80	12.60	Too low
RNA	NA	NA	10.00	11.60	12.40	Too low

Tapestation

• Only RNA samples were run on the tape station
• Due to the low sample reading from sample #4, it was not run on the tapestation

Tapestation results

Sample 1:

Sample 2:

Sample 3:

Conclusions

• Homogenates from airbrushed coral tissue can only be used for DNA, RNA is too degraded
• Need to troubleshoot homogenizing coral fragment with beadbeating, but it is possible to extract RNA using this protocol