Trying Extraction Again With RNA Purelink Kit to Get RNA from 3 Porites asteroides Samples Following Protocol from Federica

In this Extraction I used: Purelink RNA mini Kit, Trizol Reagent, 1-Bromo-3-cholropropane, and a protocol from Federica. I modified the protocol by increasing the initial volume of trizol reagent to 750ul and homogenizing with the tissuelyser for 4 minutes at 25Hz, switching tube positions at 2 minutes.

Goal: Extract high quality RNA out of these samples to send to Israel
Result: Again, no RNA extracted :(
Major Take Aways: Again, not sure why it’s not working, samples looked nicely homogenized this time. I should have added 800ul trizol at the beginning, because I was still 50ul less getting out of the beads and after the centrifugation.

Note: Protocol is done in the hood as Trizol and 1-Bromo-3-cholropropane are toxic and corrosive. PPE of lab coat, mask, eye glasses, and gloves were worn at all times. Solid and liquid wastes are hazardous.

Sample Prep

• Samples:
  • R13 A2 201907 Molec
  • R5 A2 201907 Molec
  • R14 A2 201907 Molec
• Sterilized clippers and forceps with 10% bleach, DI water, 70% ETOH, and RNase Away
• Clipped fragment on dry ice at RNA bench, brought dry ice cooler to the hood, added 750ul trizol to a glass bead tube, and used the forceps to put the chip in the bead tube
• Repeated sterilization and sample addition inside the hood for each sample
  • Tried to chip ~1cm2 of the flash frozen sample
  • Sample R14 wouldn’t chip (too thick) so I just scraped from the tissue until there was a good amount to add
• Sample R13 in bead tube:
• Sample R5 in bead tube:
• Sample R14 in bead tube:
• Put bead tubes in the tissuelyser 2 minutes at 25Hz
• Switched orientation in the tissuelyser so tubes were on the outside of the holders
• Ran the tissuelyser again for 2 minutes at 25Hz
• R13 after tissuelyser, looks like a lot if not all the tissue is off the skeleton:
• R5 after tissuelyser, also looks broken up well:
• R14 after tissuelyser, again looks well homogenized, the liquid is brown tinted:

Extraction

• Removed ~550ul from bead tubes and put into new 1.5mL tubes
• Added 450ul of Trizol to each new 1.5mL tube, vortexed tubes briefly and spun down
• Incubated tubes at room temp for 5 minutes
• Added 100ul of 1-Bromo-3-cholropropane and vortexed tubes for 15 seconds each
• The tubes became cloudy after vortexing
• Incubated tubes at room temp for 3 minutes
• Centrifuged tubes for 15 minutes at 12,000rcf at room temp
• Prepared DNase treatment on ice:
  • 24ul 10X reaction buffer
  • 30ul DNAse
  • 186ul RNase-free water
• After centrifugation tubes had phase separated:
• R13
• R5
• R14
• Was able to remove ~550ul of the top clearish layer into new 1.5mL tubes (tough to remove without getting other layers)
• Added 550ul 70% ethanol to each new tube and vortexed (note, no precipitates formed)
• Added 600ul to spin columns for each sample
• Centrifuged 25 seconds at 12,000 rcf (note, I used a different centrifuge this time that took until 20 seconds had passed to get to 12,00rcf)
• Discarded flow through in trizol waste
• Added the remaining sample to their spin columns
• Centrifuged 25 seconds at 12,000 rcf
• Discarded flow through in trizol waste
• Added 350ul of wash buffer 1
• Centrifuged 25 seconds at 12,000 rcf
• Discarded flow through in trizol waste and changed collection tubes
• Added 80ul DNase mixture to each spin column and incubated at room temp for 12 minutes
• After incubation, added 350ul wash buffer 1
• Centrifuged 25 seconds at 12,000 rcf
• Discarded flow through in trizol waste and changed collection tubes
• Added 500ul wash buffer 2
• Centrifuged 25 seconds at 12,000 rcf
• Discarded flow through in trizol waste
• Again added 500ul wash buffer 2
• Centrifuged 25 seconds at 12,000 rcf
• Discarded flow through in trizol waste
• Centrifuged columns dry for 1 minute at 12,000rcf
• Transferred columns to recovery 1.5mL tubes
• Added 100ul RNase-free water to each column
• Incubated at room temp ~1minute
• Centrifuged for 2 minutes at 16,000rcf
• Aliquoited 5ul for QC and froze the big tubes in the -80

Qubit – HS Assay this time

• Followed qubit protocol

• Did not do a tapestation