Trying Extraction Again With RNA Purelink Kit to Get RNA from 1 Porites asteroides Samples

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 grinding a large chunk on LN2, increasing the initial volume of trizol reagent to 1100ul and homogenizing with the tissuelyser for 4 minutes at 25Hz, switching tube positions at 2 minutes.

Goal: Extract high quality RNA out of one sample to get this protocol nailed down
Result: Again, no RNA extracted :(
Major Take Aways: Now I really don’t know what is the problem, maybe I have been using too much sample this whole time. That is my only guess

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

• Sample: R5 A2 201907 Molec (used all of this in the process, it was a small chip left)
• Sterilized scrapper with 10% bleach, DI water, then 70% EtOH, and RNaseZap
• Chilled mortar and pestle in -80
• Put whole chunk of sample in chilled mortar
• Poured in LN2 and ground until it was small pieces
• Scrapped pieces into a bead tube with 800ul of trizol reagent
• There was a lot of ground tissue so I added another 300ul of trizol reagent because I wasn’t sure I would be able to get out 550ul later. Total trizol volume was 1100ul
• Put the bead tube in the tissuelyser 2 minutes at 25Hz
• Switched orientation in the tissuelyser so the tube were on the outside of the holders
• Ran the tissuelyser again for 2 minutes at 25Hz
• After tissuelyser homogenization:

Extraction

• Removed 550ul from the bead tube and put into a new 1.5mL tube
• There was still more liquid in there, so I made another 1.5ml tube with 300ul of homogenized liquid. The tubes were now labeled A and B
• Increased the volume in each 1.5mL tube to 1mL with trizol reagent: 450ul for A and 700ul for B
• 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:
  • 16ul 10X reaction buffer
  • 20ul DNAse
  • 124ul RNase-free water
• After centrifugation tubes had phase separated:
• A:
• B:
• There was also a visible pellet in each tube, probably skeleton?
• A:
• B:
• 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