Trying RNA Extraction for Israel with Zymo Kit for Porites asteroides Samples

In this Extraction I used: Zymo DNA/RNA Miniprep Plus kit, and Kevin Wong’s Protocols 1 2.

Goal: Extract high quality RNA out of these 3 samples to send to Israel
Result: It worked! A good amount and good quality of RNA for each sample
Major Take Aways: Using Kevin’s protocol really worked! I may not have gotten enough RNA but it is all fairly good quality, might need to do R33 again.

Sample Preparation

• Samples:
  • R33 201907 A2 Molec
  • R13 201907 A2 Molec
  • R15 201907 A2 Molec
• Added 0.25 mL of glass beads (0.5mm) and add 500 μl of RNA/DNA shield into each empty centrifuge tube.
• Took samples out from -80 °C on dry ice.
• Used sterile clippers, added chu k tissue into the centrifuge tube with beads and RNA/DNA shield.
• Vortexed at max speed for 2 minutes.
• Removed 400 μl of the supernatant and transfer to a new centrifuge tube.
• Centrifuged at 9000 rcf for 3 minutes.
• Transferred 300 μl supernatant to a new centrifuge tube and discarded the pellet.
• Added 30 μl of Proteinase K digestion buffer (10:1 ratio of sample:digestion buffer), and 15 μl of Proteinase K (2:1 ratio of digestion buffer:Proteinase K) to each sample.
• Vortexed and spin down.
• Added 345 μl of lysis buffer.
• Continued with DNA and RNA extraction protocol

DNA Extraction

• Flicked and inverted tubes to mix and spun down
• Warmed 10mM Tris HCl pH 8 and ultra pure water in the thermomixer at 55 degrees C
• Added 700ul of the liquid to yellow spin columns and collection tubes
• Centrifuged 16,000rcf for 30 seconds
• Saved the flow through in new 5mL tubes
• Repeated addition, centrifugation, and saving of flowthrough for the remaining amount of liquid (3 total spins)
• Added 400ul DNA/RNA prep buffer to the yellow spin columns
• Centrifuged 16,000rcf for 30 seconds
• Discarded flow through (Zymo kit waste)
• Added 700ul DNA/RNA wash buffer to the yellow spin columns
• Centrifuged 16,000rcf for 30 seconds
• Discarded flow through (Zymo kit waste)
• Added 400ul DNA/RNA wash buffer to the yellow spin columns
• Centrifuged 16,000rcf for 2 minutes
• Transferred spin columns to new 1.5mL tubes for each sample labeled as final DNA tubes
• Discarded flow through and collection tubes
• Added 50ul warmed 10mM Tris HCl to the spin columns gently by dripping over the filter
• Incubated at room temp for 5 minutes
• Centrifuged 16,000rcf for 30 seconds
• Added another 50ul warmed 10mM Tris HCl to the spin columns gently by dripping over the filter
• Incubated at room temp for 5 minutes
• Centrifuged 16,000rcf for 30 seconds
• Discarded the spin columns
• Made strip tubes with 7ul of each sample in them for QC
• Kept tubes on ice bucket then stored in -20 degree freezer

RNA Extraction

• Added equal volume (1450ul) 100% ethanol to the 5mL tubes of flowthrough
• Vortexed and spun down
• Added 700ul of the liquid to green spin columns and collection tubes
• Centrifuged 16,000rcf for 30 seconds
• Discarded flow through (Zymo kit waste)
• Repeated addition, centrifugation, and saving of flowthrough for the remaining amount of liquid (5 total spins)
• Added 400ul DNA/RNA wash buffer to the green spin columns
• Centrifuged 16,000rcf for 30 seconds
• Discarded flow through (Zymo kit waste)
• Created the DNase mix:
  • 75ul DNA digestion buffer * 3 = 225ul
  • 5ul DNase I * 3 = 15ul
• Flicked and spun down mix
• Added 80ul DNAse mix to each green spin column filter
• Incubated for 15 minutes at room temp
• Added 400ul DNA/RNA prep buffer to the green spin columns
• Centrifuged 16,000rcf for 30 seconds
• Discarded flow through (Zymo kit waste)
• Added 700ul DNA/RNA wash buffer to the green spin columns
• Centrifuged 16,000rcf for 30 seconds
• Discarded flow through (Zymo kit waste)
• Added 400ul DNA/RNA wash buffer to the green spin columns
• Centrifuged 16,000rcf for 2 minutes
• Transferred spin columns to new 1.5mL tubes for each sample labeled as final RNA tubes
• Discarded flow through and collection tubes
• Added 50ul warmed ultra pure water to the spin columns gently by dripping over the filter
• Incubated at room temp for 5 minutes
• Centrifuged 16,000rcf for 30 seconds
• Added 50ul warmed ultra pure water to the spin columns gently by dripping over the filter
• Incubated at room temp for 5 minutes
• Centrifuged 16,000rcf for 30 seconds
• Discarded the spin columns
• Made strip tubes with 5ul of each sample in them for QC
• Kept tubes on ice bucket then stored in -80 degree freezer

Qubit – HS Assay

• Followed qubit protocol

Tapestation

• Followed RNA tapestation protocol
• Tapestation Report Link