Australia Mytilus Mussel Tri-Reagent RNA Extractions
Mytilus Tissue Tri-Reagent RNA Extraction
Extracting RNA from preserved Mytilus spp tissue using a Tri-Reagent/Direct-zol RNA Isolation Protocol written by Johanna Harvey.
Zymo Research Direct-zol RNA Miniprep Plus Kit
This protocol was previously tested on live M. edulis tissue with Maggie Schedl. That work can be accessed here.
Samples:
- 4590 mp TAS RNAlater
- 4596 mp TAS RNAlater
- 4598 mp TAS RNAlater
- 4599 mp TAS RNAlater
- 4593 mp TAS RNAlater
- 4597 mp TAS RNAlater
- 4611 mp TAS RNAlater
- 4512 mp TAS RNAlater
Tri-Reagent RNA Extraction
Completed on March 28, 2019
- Wear a lab coat and complete all steps that involve the Tri-Reagent and 1-Bromo-3-Chloropropane in the hood
- Aliquot 1000 uL of Tri-Reagent (stored in 4 degree fridge) per sample (8 samples total) into two 5mL tubes and keep in ice bucket
- Aliquot 100µl of 1-Bromo-3-Chloropropane (stored in the acid cabinet) per sample (8 samples total) into a 1.5mL tube and keep in ice bucket
- Clean forceps with DI Water, 70% EtOH, RNaseZap, and Nuclease-free water for each tissue sample
- Pull out pea-sized chunk of tissue from tube using the cleaned forceps, rinse in nuclease-free water and transfer to a bead tube
- Immediately add 1000 ul of Tri-Reagent to the bead tube containing the tissue piece
- Do this in the hood
- Homogenize tissue with Beadbug until tissue is fully homogenized
- Time will vary between samples
| Sample | Homogenization Time |
|---|---|
| 4590 | 120 sec |
| 4596 | 80 sec |
| 4598 | 80 sec |
| 4599 | 40 sec |
| 4593 | 160 sec |
| 4597 | 160 sec |
| 4611 | 40 sec |
| 4512 | 120 sec |
- All liquid was aspirated into a new 1.5 mL tube (don’t put on ice)
- Ended up with ~900 ul per tube
- Add 90 ul of cold 1-Bromo-3-Chloropropane to each sample tube and invert tubes repeatedly for 20 seconds (keep off ice)
- Incubate tubes at room temperature for 15 minutes
- During the incubation period, turn on centrifuge, set for 2 deg C and let spin for ~8 minutes to cool down
- After incubation, centrifuge samples at highest speed for 30 minutes
- Carefully remove tubes from centrifuge
- With a p200 ul pipette, carefully remove ~500ul of the clear supernatant (upper layer) and transfer to new 1.5 mL tube
- Put tubes containing the remaining organic phase in -80 for potential DNA extraction later
- Add equal volume (~500 ul) of 100% EtOH to the new sample tubes and invert to mix
- Transfer 500 ul of liquid into spin column from Direct-zol kit
- Centrifuge at 16000 rcf for 30 seconds at room temperature (21-22 deg C on centrifuge)
- Transfer column to new collection tube, dump out flow through and add the other 500 ul of sample repeating the step above
- After each centrifuge spin, samples are placed in new collection tubes
- Add 400 ul of RNA Wash Buffer and centrifuge at 16000 rcf for 30 seconds
- Create DNase I treatment:
- 75 ul of DNA Digestion Buffer x # of samples
- 5 ul of DNase I x # of samples
- Add 80 ul of DNase I treatment mastermix directly to the filter of the spin column
- Incubate at room temperature for 15 minutes
- DO NOT centrifuge after this step
- Begin warming nuclease-free water in the large incubator to 70 deg C
- Add 400 ul of RNA Pre-wash buffer to each spin column and centrifuge at 16000 rcf for 30 seconds
- Again, add 400 ul of RNA Pre-wash buffer to each spin column and centrifuge at 16000 rcf for 30 seconds
- Add 700 ul of RNA Wash Buffer and centrifuge at 16000 rcf for 2 minutes
- Transfer spin columns to new 1.5 ml tubes (final tubes)
- Add 50 ul of warmed nuclease-free water directly to filter of spin column
- Incubate at room temperature for 5 minutes
- After incubation, centrifuge at 16000 rcf for 30 seconds
- Again, add 50 ul of warmed nuclease-free water directly to filter of spin column
- Incubate at room temperature for 5 minutes
- After incubation, centrifuge at 16000 rcf for 30 seconds
- Aliquot 5 ul of RNA for Qubit and TapeStation analysis in strip tubes
- Store the remaining RNA in the labeled 1.5-ml tubes in the -80 freezer
Qubit BR RNA assay
Extracted RNA was quantified on the Qubit following the BR RNA assay protocol
| Sample | Avg ng/μl |
|---|---|
| Std 1 | 434 |
| Std 2 | 11110 |
| 4590 | 43 |
| 4596 | 242 |
| 4598 | 125 |
| 4599 | 122 |
| 4593 | 427 |
| 4597 | 83.8 |
| 4611 | 138 |
| 4512 | 157 |
TapeStation
Samples were also run on the TapeStation following this protocol
See full report here
__The remaining samples were completed on April 4, 2019, April 23, 2019, and May 2, 2019. The same protocol listed above was followed. Below I include the Homogenization time, Qubit BR RNA assay, and TapeStation information for the remaining samples.__
April 4, 2019
Homogenization Time
| Sample | Homogenization Time |
|---|---|
| 4385 mg FR RNAlater | 80 sec |
| 4388 mg FR RNAlater | 160 sec |
| 4395 mg FR RNAlater | 160 sec |
| 4405 mg FR RNAlater | 80 sec |
| 4425 mg FR RNAlater | 160 sec |
| 4435 mg FR RNAlater | 80 sec |
| 4445 mg FR RNAlater | 120 sec |
| 4452 mg FR RNAlater | 80 sec |
Notes from the RNA extraction process
- Sample 4405 and 4452 had about 700 ul of Tri-Reagent after re-aliquoting into 1.5 ml tubes, so I added 70 ul of the 1-Bromo-3-Chloropropane
- All the other samples had ~900 ul of Tri-Reagent, so I added 90 ul of the 1-Bromo-3-Chloropropane
- When removing the RNA phase from the remaining organic phase, sample 4405 only had ~450 ul to pull off
- Sample 4452 only had 400 ul to pull off
- All remaining samples had 500 ul to pull off
Qubit BR RNA assay
| Sample | Avg ng/μl |
|---|---|
| Std 1 | 435 |
| Std 2 | 10596 |
| 4385 | 40.3 |
| 4388 | 38 |
| 4395 | 31.8 |
| 4405 | 21.7 |
| 4425 | 60.3 |
| 4435 | 13.3 |
| 4445 | 32.3 |
| 4452 | 39.6 |
TapeStation report can be accessed here.
April 23, 2019
Homogenization Time
| Sample | Homogenization Time |
|---|---|
| Mgallo FR 4392 | 120 sec |
| Mgallo FR 4402 | 320 sec |
| Mgallo FR 4415 | 160 sec |
| Mgallo FR 4431 | 80 sec |
Notes from the RNA extraction process
- Samples 4415 and 4431 had less tissue than the other samples
- All samples had about 900 ul of Tri-Reagent after re-aliquoting into 1.5 ml tubes, so I added 90 ul of the 1-Bromo-3-Chloropropane
Qubit BR RNA assay
| Sample | Avg ng/μl |
|---|---|
| Std 1 | 419 |
| Std 2 | 10756 |
| 4392 | 47.4 |
| 4402 | 75.6 |
| 4415 | 65 |
| 4431 | 26.9 |
TapeStation report can be accessed here.
May 2, 2019
Homogenization Time
| Sample | Homogenization Time |
|---|---|
| Mgallo MN 4428 | 200 sec |
| Mgallo Primel 2 | 244 sec |
| Mgallo Primel 6 | 240 sec |
| Mgallo Primel 9 | 200 sec |
Notes from the RNA extraction process
- Sample 4428 had less tissue than the other samples
- Sample Primel 9 had its tissue piece stuck to the lid of the tube, so it may not have been preserved in RNase
- All samples had about 950 ul of Tri-Reagent after re-aliquoting into 1.5 ml tubes, so I added 95 ul of the 1-Bromo-3-Chloropropane
Qubit BR RNA assay
| Sample | Avg ng/μl |
|---|---|
| Std 1 | 417 |
| Std 2 | 10806 |
| 4428 | 25 |
| Primel 2 | 194 |
| Primel 6 | 49.9 |
| Primel 9 | 76.7 |
TapeStation report can be accessed here.
Once RNA was extracted from all the samples, Maggie Schedl and I performed the probe synthesis steps from the EecSeq protocol to create sequence capture probes.
The probe synthesis steps are documented here:
Written on January 22, 2021