Using Zymo Quick-RNA Miniprep Plus Kit

Extracting RNA from juvenile oyster samples from multi-stressor exposure experiment.

Juveniles were collected individually in 1.5ml tubes, preserved with liquid nitrogen, and stored in -80 freezer until processing.

This is the general protocol I followed for RNA Extractions adapted from dual DNA/RNA extraction protcol. I typically processed 10-12 samples at a time.

Time to Completion: 5-6 hours (includes QC)

Prep tubes

Prep and label all tubes for extraction protocol (for each tube type, multiply by number of samples being processed)

tube	purpose	number
1.5 mL	homogenization/digest tube	* (# of samples)
1.5 mL	intmed. DNA steps	* (# of samples)
1.5 mL	intmed. RNA steps	* (# of samples)
Yellow spin-column & collection tubes	Spin-away DNA	* (# of samples)
Green or white spin-column & collection tubes	RNA purification	* (# of samples)
1.5 mL	final RNA sample	* (# of samples)

Homogenization

• Turn on thermomixer and set to 55 degrees C, speed 1000 rpm
• Pull Proteinase K out of -20 freezer to warm to room temperature
• Pull DNAse I out of -20 freezer to thaw on ice
• In homogenization/digest tubes, add 300 μL of DNA/RNA Shield
• Pull samples out of -80 freezer and put on ice
• Sterilize forceps with 100% EtOH, Type II DI Water, RNAse Zap, and RNAse-free Water
• Sterilize forceps between each sample
• Using forceps, transfer juvenile to 1.5 mL tube with DNA/RNA Shield
• Sterilize cone-shaped dremmel bit with 100% EtOH, Type II DI Water, RNAse Zap, and RNAse-free Water
• Sterilize the dremmel bit between each sample
• Insert dremmel bit into 1.5 mL tube with juvenile and grind up juvenile for ~10 seconds at speed 5
• Add 30 μL PK Digestion Buffer to each tube
• Add 15 μL Proteinase K
• Vortex and spin down
• Incubate in thermomixer at 55 degrees C and 1000 rpm for 30 minutes
• Halfway through incubation, spin down tubes in tabletop centrifuge for 1 minute at 13000 rpm

During incubation period:

• Pull Biotium RNA kit out of fridge/freezer and put in drawer (light sensitive) to warm to room temperature
• Prep RNAse-free Water for thermomixer
  • 50 μL x # of samples (plus error) in 1.5 mL tubes
  • Put in thermomixer set to 70 degrees C
• Make DNAse reaction mix:
  • DNA Digestion Buffer: 75 μL x # of samples
  • DNAse I: 5 μL x # of samples

After incubation:

• Spin down tubes at max speed for 2 minutes in tabletop centrifuge
• Without disturbing the debris pellet, gently transfer all supernatant (~350 μL) to new 1.5 mL tube for DNA removal (intmed. DNA steps)
• Save debris pellet and store in -20 freezer (can check for incomplete digestion later on if necessary)
• Add equal volume (300 μL) of RNA Lysis Buffer (kit) to each tube
• Finger flick to mix
• Transfer all supernatant (~600 μL) to yellow spin-column
• Centrigue at 16000 rpm for 30 seconds
• SAVE flow-through in new 1.5 mL tube for RNA

RNA Purification

• Add equal volume (600 μL) 100% EtOH to each 1.5 mL tube for RNA
• Transfer 600 μL of supernatant to white spin-column
• Centrifuge at 16000 rpm for 30 seconds; discard flow-through
• Repeat until all supernatant is filtered (2 times total)
• Add 400 μL of RNA Wash Buffer (kit) to white spin-column
• Centrifuge at 16000 rpm for 30 seconds; discard flow-through
• Add 80 μL of DNAse reaction mix to each column (I add directly onto filter)
• Incubate at room temperature for 30 minutes
• NOW IS A GOOD TIME TO PULL OUT RNA QUBIT STANDARD AND TAPESTATION SUPPLIES
• Add 400 μL of RNA Prep Buffer (kit) to white spin-column
• Centrifuge at 16000 rpm for 30 seconds; discard flow-through
• Add 700 μL of RNA Wash Buffer (kit)
• Centrifuge at 16000 rpm for 30 seconds; discard flow-through
• Add 400 μL of RNA Wash Buffer (kit)
• Centrifuge at 16000 rpm for 2 minutes; discard flow-through
• Transfer spin-column to final RNA 1.5 mL tube
• Carefully drip 50 μL of warmed RNAse-free Water directly onto filter
• Incubate at room temperature for 5 minutes
• Centrifuge at 16000 rpm for 30 seconds and discard spin column
  • KEEP FLOW-THROUGH
• Should have 50 μL of RNA

Quality Control Check

Qubit

• RNA concentrations are checked using the Biotium RNA Broad Range quantitation kits
• use 1 μL of sample for each of the assays (unless you need to use more)
• Final RNA concentrations (ng/μL) can be accessed here

TapeStation

• RNA quality is checked using the RNA ScreenTape Assay
• Sample TapeStation report can be accessed here