Sep 29, 2023

Public workspaceSanger Tree of Life RNA Extraction: Automated MagMax™ mirVana

DOI
dx.doi.org/10.17504/protocols.io.6qpvr36n3vmk/v1
  • 1Tree of Life, Wellcome Sanger Institute, Hinxton, Cambridgeshire, CB10 1SA;
  • 2Wellcome Sanger Institute
Tree of Life Genome Note Editor
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DOI: dx.doi.org/10.17504/protocols.io.6qpvr36n3vmk/v1
Protocol Citation: Raquel Juliana Vionette do Amaral, Amy Denton, Halyna Yatsenko, Jessie Jay, Caroline Howard, Adam Bates 2023. Sanger Tree of Life RNA Extraction: Automated MagMax™ mirVana. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr36n3vmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 31, 2023
Last Modified: September 29, 2023
Protocol Integer ID: 85710
Keywords: RNA extraction, automated RNA extraction, magnetic beads, MagMax, mirVana, reference genome
Funders Acknowledgement:
Wellcome Trust
Grant ID: 218328
Wellcome Trust
Grant ID: 206194
Gordon and Betty Moore Foundation
Grant ID: GBMF8897
Abstract
This protocol describes the automated extraction of RNA from multiple different tissue samples intended for RNA-Seq using the MagMax™ mirVana total RNA isolation kit and the Thermo Fisher KingFisher™ Apex. This process is highly effective for the majority of taxonomic groups covered by the Tree of Life Programme, however, challenging samples include corals, jellyfish and annelids. The output of this protocol is a highly concentrated RNA extract which can be diluted and submitted for RNA-Seq on Illumina NovaSeq.
Guidelines
  • All steps can be performed at room temperature unless stated otherwise.
  • If samples are not going to proceed to sample lysis immediately, keep samples on dry ice to maintain temperature and prevent nucleic acid degradation.
  • An experienced operator can expect to comfortably process up to 32 samples, with approximately 2 hours handling time over a start to finish period of 3 hours. This estimation excludes subsequent QC checks.

Additional Notes:
  • FluidX tubes are used throughout the Tree of Life programme in order to track samples, therefore rather than the microcentrifuge tubes which have been mentioned in this protocol for RNA storage, all routine RNA extracts are stored in FluidX tubes.
  • Both the KingFisher™ Apex protocol script and the KFX.file have been made available for this protocol - the KFX.file requires ‘BindIx software for KingFisher Apex’ to allow the KingFisher™ Apex protocol to be viewed on a PC or laptop. Alternatively, the file can be transferred directly onto a KingFisher™ Apex instrument using a USB.
Materials
  • MagMAX mirVana Total RNA isolation kit (Boxes 1 & 2, Box 1 should be stored in the freezer and Box 2 at room temperature) (Thermo Fisher Cat. no. A27828)
  • Thermo Fisher KingFisher™ 1 mL 96-well Deep-well Plates (Thermo Fisher Cat. no. 95040450)
  • Thermo Fisher KingFisher™ 96 Deep-well Tip Comb (Thermo Fisher Cat. no. 97002570)
  • Thermo Fisher KingFisher™ 200 µL standard 96-well Plate (Thermo Fisher Cat. no. 97002084)
  • 1.5 mL BioMasher tubes and pestles (sterile) (Cat. no. 9791A)
  • 2 mL DNA Lo-Bind microcentrifuge tubes (Eppendorf Cat. no. 0030 108.078)
  • 15 mL or 50 mL centrifuge tubes
  • Dry ice
  • Ice
  • 100% absolute ethanol
  • 100% absolute isopropanol
  • Dithiothreitol (DTT) (Cat. no A3668.0050)

Equipment
  • Pipettes for 0.5 to 1000 μL and filtered tips
  • Diagnocine PowerMasher II tissue disruptor (Cat. no. FNK-891300)
  • Vortexer (Vortex Genie™ 2 SI-0266)
  • Plate shaker/Thermomix (Thermo Fisher,Cat. no. 88882006)
  • Thermo Fisher KingFisher™ Apex instrument (Cat. no. 5400930)



KingFisher™ Apex RNA Extraction Protocol Script:

KFX File: Download MagMax mirVana.kfxMagMax mirVana.kfx2KB

1. Pick Up Tip - Tip Plate
2. Lysis & Bind - Sample Plate
Pre-collect beads: Off
Release beads: On 00:00:00
Heating & Cooling: Off
Mixing 1# 00:07:00 Medium
Postmix: Off
Collect beads: On 5 Count 2 Seconds
3. Wash 1 - Wash Plate 1
Pre-collect beads: Off
Release beads: On 00:00:00
Heating & Cooling: On 4°C Preheat: Off
Mixing 1# 00:01:00 Fast
Postmix: Off
Collect beads: On 5 Count 1 Second
4. Wash 2 - Wash Plate 2
Pre-collect beads: Off
Release beads: On 00:00:00
Heating & Cooling: On 4°C
Preheat: Off
Mixing 1# 00:01:00 Fast
Postmix: Off
Collect beads: On 5 Count 1 Second
5. Dry 1 - Wash Plate 2
Duration: 00:02:00 Dry Type: Outside Well
6. DNase Step - DNase Plate
Pre-collect beads: Off
Release beads: On 00:00:05 Bottom mix
Heating & Cooling: Off
Mixing 1# 00:15:00 Medium
Postmix: Off
Collect beads: Off
7. Dispense - DNase Plate
Custom naming: Add 50µl Rebinding Buffer + 100 µl Isopropanol
Dispense to plate: Isopropanol 100 µl
Rebinding Buffer 50 µl
8. Rebinding - DNase Plate
Pre-collect beads: Off
Release beads: On 00:00:05
Heating & Cooling: Off
Mixing 1# 00:05:00 Medium
Postmix: Off
Collect beads: On 5 Count 1 Second
9. Wash 3 - Wash Plate 3
Pre-collect beads: Off
Release beads: On 00:00:00
Heating & Cooling: On 4°C Preheat: Off
Mixing 1# 00:01:00 Fast
Postmix: Off
Collect beads: On 5 Count 1 Second
10. Wash 4 - Wash Plate 4
Pre-collect beads: Off
Release beads: On 00:00:00
Heating & Cooling: On 4°C Preheat: Off
Mixing 1# 00:01:00 Fast
Postmix: Off
Collect beads: On 5 Count 1 Second
11. Dry 2 - Wash Plate 4
Duration: 00:02:00 Dry Type: Outside Well
12. Elute - Elution Plate
Pre-collect beads: Off
Release beads: Off
Heating & Cooling: On 60°C Pre-heat: On
Mixing 1# 00:05:00 Medium
Postmix: On 00:00:05 Fast
Collect beads: On 5 Count 4 Seconds
13. Leave Tip - Tip Plate


Protocol PDF: Download Sanger Tree of Life RNA Extraction_ Automated MagMax MirVana.pdfSanger Tree of Life RNA Extraction_ Automated MagMax MirVana.pdf89KB

Safety warnings
  • The operator must wear a lab coat, powder-free nitrile gloves and safety specs to perform the laboratory procedures in this protocol. Cotton glove liners are strongly recommended when handling the samples on dry ice.
  • Waste needs to be collected in a suitable container (e.g. plastic screw-top jar or Biobin) and disposed in accordance with local regulations.
  • Liquid waste needs to be collected in a suitable container (e.g. glass screw-top jar) and disposed in accordance with local regulations.
  • Do not open the door of the KingFisher™ Apex instrument whilst it is in operation.
Before start
  • Add 10 mL of absolute isopropanol to Wash solution 1, mix well and store at room temperature.
  • Add 48 mL of absolute ethanol to Wash solution 2, mix well and store at room temperature.
Reagent Preparation
Reagent Preparation
Prepare the TURBO DNase solution as described below, and once made, store on wet ice:
ComponentVolume per sample (µL)
Turbo DNase (stored in freezer)2.5
MagMAX TURBO DNase Buffer60

Prepare the Binding Beads Mix as described below, and once made, store on wet ice:
ComponentVolume per sample (µL)
RNA binding beads12
Lysis/binding enhancer (stored in freezer)12

Sample Lysis
Sample Lysis
Calculate the amount of lysis buffer required for the samples – 40 µL is required per 1 mg of tissue. 15 mg of sample is used for this protocol, so 600 µL per sample is required.
Create sufficient lysis buffer for your samples:
ComponentVolume per sample (µL)
Lysis Buffer1000
DTT0.7

For samples that require powermashing, transfer 15 mg of tissue into a 1.5 mL BioMasher II tube and add 600 µL of lysis buffer. Disrupt sample in the lysis buffer using a PowerMasher II tissue disruptor and the BioMasher pestle, until no large pieces remain or sample cannot be disrupted further (for more detailed instructions regarding powermashing, please refer to the Sanger Tree of Life Sample Homogenisation: Powermash protocol).
For samples that have been cryoprepped, transfer the 15 mg of cryoprepped tissue into a 2 mL microcentrifuge tube and add 600 µL of lysis buffer. Pipette mix to homogenise the cryoprepped tissue and lysis buffer.
Incubate at room temperature for 30 seconds to 1 minute to allow samples to lyse. If samples will not immediately progress to Step 8, place samples on ice until ready to proceed.
Loading and Running the KingFisher™ Apex
Loading and Running the KingFisher™ Apex
Transfer 200 µL of each sample directly into individual wells of a Thermo Fisher KingFisher™ 1 mL 96-well deep-well plate.
Add 100 µL of isopropanol to each sample, seal the plate and shake at room temperature on a plate shaker for 2 minutes at 950 rpm.
Add 20 µL of the prepared binding beads mix to each sample, re-seal the plate and mix at room temperature on a plate shaker for 5 minutes at 950 rpm.
Prepare the remaining processing plates for the KingFisher™ Apex protocol:
Plate IDPlate positionPlate typeReagents(s) requiredVolume per well
Sample Plate1Deep-wellSample + isopropanol + binding beads mix200 µL sample + 100 µL isopropanol + 20 µL binding beads mix
Wash Plate 12Deep-wellWash solution 1150 µL
Wash Plate 23Deep-wellWash solution 1150 µL
DNase Plate4Deep-wellTURBO DNase solution50 µL
Wash Plate 35Deep-wellWash solution 2150 µL
Wash Plate 46Deep-wellWash solution 2150 µL
Elution plate7Standard (200 µL)Elution buffer50 µL
Tip Comb8Deep-wellPlace a tip comb in the plate


On the KingFisher™ Apex, select the protocol (details below in the KingFisher™ Apex RNA Extraction Protocol Script/attached KFX file in the Materials Section) on the protocols list and select using the play button.
Load the processing plates and the sample plate in the positions prompted by the instrument and then start the run. The full protocol will take approximately 50 minutes.
After 30 minutes, the protocol will pause and there will be a prompt to remove the DNase plate from the instrument, and add 50 µL of the rebinding buffer and 100 µL absolute isopropanol to each well containing sample as quickly as possible – do not premix these reagents and always add them separately to the wells.
Load the DNAse plate back into the instrument and press run to resume the protocol.
At the end of the run, remove the elution plate and store on ice.
Inspect the elution plates for any magnetic beads in the wells. In the rare instance of magnetic beads remaining in the eluate (possible in viscous samples), these samples will need to be transferred to a 1.5 mL microcentrifuge tube and placed on a magnetic rack. Allow around 5 minutes for the beads to migrate and take the clear eluate containing the RNA using a pipette tip.
Pipette the eluates into microcentrifuge tubes, perform the required QC, and then store eluates at
–80°C.
Protocol references
Applied Biosystems MagMAX™ mirVana™ Total RNA Isolation Kit User Guide: Document Connect (thermofisher.com)