Nov 01, 2024
Version 2
Purification of viral RNA from cell culture isolates or FTA cards using MagMAX Viral RNA Isolation Kit V.2
- Erika Bujaki1,
- Dimitra Klapsa1,
- Kafayat Arowolo1,
- Joyce Akello2,
- Nick Grassly2,
- Javier Martin1
- 1MHRA;
- 2Imperial College London
- Poliovirus Sequencing Consortium
Protocol Citation: Erika Bujaki, Dimitra Klapsa, Kafayat Arowolo, Joyce Akello, Nick Grassly, Javier Martin 2024. Purification of viral RNA from cell culture isolates or FTA cards using MagMAX Viral RNA Isolation Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v928mml3e/v2Version created by Erika Bujaki
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: March 26, 2024
Last Modified: November 01, 2024
Protocol Integer ID: 111406
Funders Acknowledgement:
Bill and Melinda Gates Foundation
Grant ID: INV-043859
Medicines and Healthcare product Regulatory Agency
Abstract
This protocol describes the viral nucleic acid recovery and purification from poliovirus isolates either in their original liquid form or spotted onto FTA cards. The method utilises the magnetic bead based MagMAX ‱ Viral RNA Isolation Kit. When using isolates directly, progress to one of the extraction workflows straight after reagent preparation, skipping Section 2. To process FTA card discs follow the procedures in Sections 1 and 2 in full before extraction. To perform manual extractions follow Workflow A, for automated extraction on King Fisher Duo Prime follow steps in Workflow B, and for automated extraction on King Fisher Flex follow Workflow C.
Version 2 has minor edits to a few steps and the King Fisher programs attached.
Materials
Reagents required specifically for FTA card processing
- TE buffer (10mM TRIS-HCl/0.1mM EDTA pH 8) (Thermo Fisher, Cat. No. 12090-015)
- Glycogen, Nuclease Free (Ambion Cat. No. AM9510)
- DTT (Dithiothreitol powder, Sigma-Aldrich Cat. No. D-9779 or 1M solution in ready to use, sterile formulation)
- Nuclease free water (for preparing 1M DTT solution)
Reagents required for RNA extraction
- RNaseZap RNase Decontamination Solution (Thermo Fisher, Cat. No. AM9780)
- Ethanol (96-100%)
- Isopropanol (100%)
- MagMAX Viral RNA Isolation Kit (AM1939). Contains sufficient reagents to isolate RNA from approximately 65 samples (see table below)
- Individual components of the kit can also be purchased separately in larger volumes (as shown with product codes in table below)
| A | B | C | D | |
| Amount | Component | Product code | Storage | |
| 100 mL | MagMAX Lysis/Binding Solution Concentrate | AM8500 | Room temp | |
| 205 mL final volume | Wash Solution 1 Concentrate (Add 70mL 100% isopropanol before use) | AM8504 | Room temp | |
| 200 mL final volume | Wash Solution 2 Concentrate (Add 160 mL 100% Ethanol before use) | AM8640 | Room temp | |
| 10 mL | MagMAX Total RNA Elution Buffer | A41043 | Room temp | |
| 500 µL | Carrier RNA | 4382878 | -20 °C | |
| 1.8 mL | DNA Binding Beads (same as RNA binding beads in AM1939 kit) | 4489112 | 4 °C | |
| 5 x 1.25 mL | Recombinant Proteinase K Solution (20 mg/mL) (Same as Lysis/Binding Enhancer in AM1939 kit) | AM2548 | -20 °C |
Equipment
- Biological Safety Cabinet II (Class II BSC)
- PPE
- Magnetic stand accommodating 1.5-2 mL centrifuge tubes / King Fisher Duo Prime equipped with 12 tip-magnetic head / King Fisher Flex equipped with 96 tip deep well magnetic head
- Pipettes and sterile, filtered, RNAse-free pipette tips
- 1.5 mL microcentrifuge tubes (the use of 1.5 mL Eppendorf DNA LoBind tubes or Processing Tubes included in the extraction kit is recommended)
- 15 mL / 50 mL centrifuge tubes for preparing Lysis/Binding Solution
- Tube racks
- Themomixer or heating block accommodating 1.5 mL/ 2mL microcentrifuge tubes incubating at 70 °C
- Vortex/shaker/rotator/thermomixer accommodating 1.5-2 mL centrifuge tubes
- 4mm Disposable Biopsy Punch with plunger (Miltex, Northumbrian Medical Supplies, Cat. No. 33-34-P/25 or Mediworld, Cat. No. 226M36)
- Cutting Mat (Qiagen Cat.No. WB100020)
- Sharps Waste container to dispose used biopsy punchers
- 0.45µm syringe filter with sterile 5 mL syringe for sterile filtration of 1M DTT if prepared from powder
- Centrifuge accommodating 1.5-2 mL tubes
- Plasticware for the use of King Fisher Duo Prime equipment with 12-tip magnet, as shown in Workflow B
- Plasticware for the use of King Fisher Flex equipment with 96-tip magnetic head, as shown in Workflow C
Reagent Preparation
Reagent Preparation
15m
15m
To process samples from FTA cards, prepare all the reagents listed, to start from liquid cell culture isolates prepare reagents in steps 1.3-1.6 only.
DTT solution (1M)
- Dissolve 0.6168g of DTT in 4 ml of Nuclease Free water.
- Sterilize by filtration with 0.45 µm syringe filter unit and a 5 ml syringe.
- Prepare aliquots and store at -20°C.
5m
RNA Processing Buffer
Combine the components below to prepare enough RNA Processing Buffer for the required number of samples and store on ice. RNA Processing Buffer can also be made ahead of time and stored at -20 °C
for up to six months.
| A | B | |
| Component | Volume for 12 samples | |
| Glycogen (5mg/ml) | 160 μl | |
| 1M DTT | 8 μl | |
| TE-1 | 3832 μl | |
| Total volume | 4000 μl |
3m
Wash Solution 1
- Add indicated volume of 100% Isopropanol to the bottle of Wash Solution 1 Concentrate.
- Mix well by inverting at least 5 times and mark bottle to indicate that the alcohol was added.
1m
Wash Solution 2
- Add indicated volume of 100% ethanol to the bottle of Wash Solution 2 Concentrate.
- Mix well by inverting at least 5 times and mark the bottle to indicate that ethanol was added.
Note
Taking an aliquot of the prepared Wash Solutions is recommended to avoid potential contamination if they are to be used on multiple occasions.
1m
Lysis/Binding Solution
Defrost Carrier RNA on ice and combine the components listed below in the order indicated. Prepare enough reagents for the number of samples extracted that day, including controls, with adding extra 10% for pipetting loss.
- Add Carrier RNA to Lysis/Binding Solution Concentrate according to the table below, and mix briefly
| A | B | |
| Reagent | Per sample | |
| Lysis/Binding Soln. Concentrate | 200 μl | |
| Carrier RNA | 1 μl |
- Add 100% isopropanol and mix well by vortexing.
| A | B | |
| Component | Per sample | |
| 100% Isopropanol | 200 μl |
Note
Prepare the Lysis/Binding Solution on the day it will be used. Keep at room temperature with a tightened lid
3m
Bead Mix
- Vortex the nucleic acid binding beads well to ensure that the beads are fully resuspended.
- Combine the components that are listed below:
| A | B | |
| Component | Volume per sample | |
| Nucleic Acid Binding Beads | 7 μl | |
| Lysis/Binding ENHANCER | 7 μl | |
| Total volume | 14 μl |
- Mix well by vortexing and place the prepared bead mix on top of ice or in the fridge until it is needed, but avoid freezing as it destroys its properties.
Note
Prepare the bead mix on the day it will be used. Bead mix can be stored on ice until it is needed for up to 4 hours. Avoid freezing the mixture as it damages the magnetic beads.
2m
FTA card Processing
FTA card Processing
22m
22m
Follow the below steps if using FTA cards. For liquid isolates, progress to RNA extraction section following workflow A/B/C depending on the mode of extraction.
Set the thermal block to 70°C.
For each sample:
Set up and label clean nuclease free 1.5 mL microcentrifuge tubes for each FTA card to be processed.
2m
Open one FTA card pouch and take out the card. Place the cutting mat under the FTA card marked area, where the paper will be punched.
Using a new 4mm disposable punch, punch out seven (7) punches per sample and place in the labelled 1.5 ml microtube. (Do not punch too hard or the punch will dull and not cut).
Note
Avoid moving the FTA card around on the cutting mat, so as not to contaminate the cutting surface.
Note
Ensure FTA card is punched in a manner to allow for additional round of extraction if necessary with positioning the punched holes accordingly.
5m
Dispose of the biopsy punch in the sharps bin and use new punch for each card.
Place the next card in a different spot on the cutting mat. The front and back of the mat may be used before having to clean the mat. (Do not use the same spot as before to punch a new card to avoid contamination between cards).
Aliquot 300 μl of RNA Processing Buffer into each tube with the FTA card discs.
2m
Close and vortex tubes at high speed for 40 seconds.
5m
Incubate tubes at 70°C for 5 minutes at 800 rpm speed. (If not using thermomixer, take tubes and mix by vortexing briefly at moderate speed every 2 minutes to a total of 3 times.)
Note
Ensure the tube lids are properly closed during incubation to prevent popping open during mixing.
6m
When incubation is over, check the lids are closed properly, vortex and centrifuge tubes briefly to collect content. (You will be able to recover up to ~240 µl of supernatant for RNA extraction from each sample).
Process sample supernatants to extract viral RNA by following one of the extraction workflows in step 3/4/5.
2m
Manual extraction - Workflow A
Manual extraction - Workflow A
7h 8m
7h 8m
The indicated processing times are based on handling 12 samples at the same time .
1h 10m
Prepare the lysate:
For each sample:
- Set up and label 1.5 ml Eppendorf DNA LoBind centrifuge tubes, then aliquot 400 μl of the lysis/binding solution (supplemented with carrier RNA and 100% Isopropanol - see Reagent preparation section) into each tube.
2. Transfer 200 μl of the liquid isolate or the full recoverable volume from the processed FTA card supernatant to the labelled tubes containing the Lysis/Binding Solution (supplemented with carrier RNA and 100% Isopropanol).
Note
When adding sample, check and confirm the sample ID. Immerse pipette tips slightly in the Lysis/Binding Solution to prevent creating aerosols and rinse pipette tip. Keep all other tubes closed to avoid cross contamination.
3. Mix gently by vortexing for 30s and spin briefly to collect tube content.
10m
Bead capture and washes:
4. Add 14 μl of prepared bead mix to each sample tube containing the lysed sample solution.
Note
Mix bead solution by pipetting to avoid settling down of the magnetic beads. Use a new tip for each addition to minimise bead loss and rinse the tip gently a few times to ensure full volume is transferred.
5. Mix tubes thoroughly at gentle speed for 4 min to fully lyse viruses and bind RNA to beads.
Note
A shaker or rotator mixer can be used for this step or tubes can be repeatedly rotated or gently vortexed manually. It is important to achieve sufficient mixing in this step with a visibly homogenous coloured mixture throughout to ensure efficient lysis and bead binding.
6. Centrifuge tubes briefly to collect content, then place on magnet and leave for at least 2 minutes to allow for bead capture to complete. Beads should form a pellet against the magnet.
Note
Pellets may also be smeared on the tube’s wall as well as forming a compact body. Twisting the tube gently help to make the pellet more compact if needed.
7. Carefully aspirate and discard supernatant without disturbing the beads.
8. It is important to remove the lysis supernatant fully, so a brief centrifugation before collecting the remaining supernatant might be necessary.
9. Remove tubes from magnetic stand and place in tube rack for washing with Wash Solution 1.
10. Add 200 µl Wash Solution 1 to each sample and vortex at moderate speed for 30s.
Note
Pellets do not necessarily get fully resuspended in all samples.
11. Centrifuge briefly to collect tube content.
12. Capture beads on magnet until mixture becomes clear, indicating full capture.
13. Carefully aspirate and discard supernatant.
14. Repeat steps 9-13 one more time to complete two washes with Wash Solution 1.
15. Remove tube from magnetic stand and place in tube rack for washing with Wash Solution 2.
16. Add 300 µl Wash Solution 2 to each sample and vortex at moderate speed for 30s.
Note
Beads can appear granular during washing steps with Wash Solution 2.
17. Centrifuge briefly to collect tube content.
18. Capture beads on magnet for 2 mins or until mixture becomes clear, indicating full capture.
19. Carefully aspirate and discard supernatant.
20. Repeat steps 15-19 to complete two washes with Wash Solution 2.
Note
It is important to completely remove the supernatant after the second wash to avoid inhibition in downstream applications.
50m
Drying the beads and elution:
21. Centrifuge briefly and remove any residual solution with a small volume fine-tipped pipette, without disturbing the pellet.
22. Dry the beads by leaving the tube open for 2 minutes to allow any remaining alcohol to evaporate.
Note
Avoid overdrying the beads to the point of the pellet cracking and crumbling as this may lower the efficiency of nucleic acid recovery.
23. Add 50 µL Elution Buffer to each sample and shake/vortex vigorously for 4 min to fully resuspend the pellet.
24. Centrifuge briefly to collect tube content.
25. Capture the beads on the magnet as before and collect supernatant containing the purified RNA in labelled containers and keep on ice for immediate use or store frozen until needed.
Note
Open tubes before placing on the magnet to avoid tube content flipping on sidewall. When this happens, remove tube and centrifuge briefly before replacing on the magnet.
Note
If beads are accidentally collected, return fluid from the pipette tip, and try again once supernatant is fully clear.
Note
Pellet might be smeared around the bottom of the tube instead of forming a compact pellet against the magnet. Look into the tube from above to check if eluate has fully cleared. Aim to collect RNA from a central position without touching the pellet.
10m
Automated extraction using a King Fisher Duo Prime - Workflow B
Automated extraction using a King Fisher Duo Prime - Workflow B
7h 8m
7h 8m
MVRI_DUO_SV_300ul.bdz3KB Read the King Fisher Duo Prime instrument manual for installation and operating instructions in its entirety before operating the magnetic particle processor. Import the attached MVRI_Duo_SV_300ul program and run a mock test.
Up to 12 samples can be extracted at the same time in one plate using the 12-tip magnetic head.
Plasticware for small volume sample extractions on King Fisher Duo Prime equipped with 12-tip magnetic head.
| A | B | |
| Item | ThermoFisher product code | |
| KingFisher deep-well 96 plate (50) | 95040450 | |
| King Fisher Duo Combi Pack for 96 DW Plate All plasticware for extraction of 8 plates (96samples) | 97003530 | |
| KingFisher Duo cap for elution strip (40) | 97003540 | |
| KingFisher Duo elution strip (40) | 97003520 | |
| KingFisher 96 KF plate | 97002540 | |
| KingFisher 12 tip comb for 96 deep-well plate (50) | 97003500 |
54m
Preparing 96 deep well plate:
Note
Label your plates and elution strips if using more than one.
The clean reagents should be added to the plate in a separate dedicated area, away from where samples are handled to avoid contamination.
Set up plate by loading the required reagents into the appropriate positions as shown in table below:
1. Aliquot 400 μl of the Lysis/Binding Solution supplemented with carrier RNA and 100% Isopropanol (see step 1 and 2 in Reagent preparation) to the top row (row A) of the 96 deep-well plate.
2. Add 200 μl of prepared wash solution 1 to rows B and C of the 96 deep well plate.
3. Add 300 μl of prepared wash solution 2 to rows D and E of the 96 deep well plate.
4. Place a 12-tip comb in a 96 deep well plate in Row H.
5. Add 60 μl of Elution buffer/ nuclease free water to the elution strip tube.
6. Move the plate to the sample handling area and transfer 200 μl of the liquid isolate or all the recovered volume (200-240 μl) of the processed FTA card supernatant to the same top row (row A) of the 96 deep-well plate containing the Lysis/Binding Solution. Mix by gently pipetting up and down in the 96 deep-well plate a few times. (Discard tube containing the punches).
Note
When adding sample, check and confirm the sample ID. Immerse pipette tips slightly in the lysis/binding solution to prevent creating aerosols and rinse pipette tip. Keep all other tubes closed to avoid cross contamination.
7. Add 14 μl of prepared bead mix to row A of the 96 deep well plate containing the lysed sample solution using a new tip for every addition and rinsing it with the sample solution.
Note
Mix bead solution by pipetting to avoid settling down of the magnetic beads. Use a new tip for each addition and rinse the tip gently a few times to ensure full volume is transferred.
20m
Setting up and running the King Fisher Duo Prime:
8. Check to confirm that the KingFisher Duo Prime is set up with 12-tip magnet and heating block.
Follow the below steps to change the magnetic
head on the King Fisher Duo Prime if necessary:
- Select and start Change Magnetic Head protocol in Maintenance protocols in the device menu. (This will position the magnet to be accessible.)
- Unscrew and remove the screws holding the magnetic head in place and lift the magnetic head to take it out.
- Replace the required magnetic head and tighten the screws to hold it in place.
- Remember to also change the heating blocks as the machine doesn’t give a prompt to do so!
- Run Check 12 tip protocol with a dummy test plate containing the tip comb in the required row to ensure the right positioning. Unload the test plate.
9. Load the prepared sample plate onto the King Fisher Duo Prime as prompted, ensuring the right orientation by matching the A1 marking on the turntable. Ensure that the plate is lying completely flat.
10. Place the prepared elution strip into the device in the metal rack next to the loaded plate and use the fold over lock on the elution block to secure it in place. Use the position of the small round hole on the elution strip to match the red conical protrusion on the rack to ensure the right orientation. Both the plate and the elution strip should be on the same side of the turntable.
11. Select the MVRI_DUO_SV_300ul Protocol and press start.
12. Close the front lid while the KingFisher is running.
31m
Un-loading the device:
13. After completion of the run, a final prompt will appear. “Unload RNA plate and RNA Elution Strip”.
14. Unload the elution strip containing the RNA, cap and place the elution strip containing the RNA on ice.
15. Remove the sample plate from the device then press the “Check Mark”. Discard the plate and content following the appropriate laboratory procedure. Wipe clean equipment, then switch on UV light for disinfection.
16. Transfer eluted viral RNA to labelled containers and keep on ice for immediate use or store frozen until needed.
3m
Automated extraction using a King Fisher Flex - Workflow C
Automated extraction using a King Fisher Flex - Workflow C
7h 8m
7h 8m
MVRI_Flex_SV_300ul.bdz3KB Read the King Fisher Flex instrument manual for installation and operating instructions in its entirety before operating the magnetic particle processor. Import the attached MVRI_Flex_SV_300ul program and run a mock test.
Up to 96 samples can be extracted at the same time in one plate using the 96-tip magnetic head.
Plasticware for small volume sample extractions on King Fisher Flex equipped with 96-tip magnetic head.
| A | B | |
| Item | ThermoFisher product code | |
| KingFisher deep-well 96 plate (50) | 95040450 | |
| KingFisher 96 tip comb for deep-well magnets (100) | 97002534 | |
| KingFisher 96 KF plate For tip comb placement and eluate storage (48) | 97002540 |
1h 30m
Setting up the processing plates
The clean reagents should be added to the plate in a separate dedicated area, away from where samples are handled to avoid contamination..
1. Label and prepare the processing plates according to the table below:
Note
Using an automated multidispenser pipette with appropriate combitips tips or the use of multichannel pipettes is recommended for the following steps for maintaining accuracy with solutions containing alcohols and to reduce repetitive strain. Adjust volume and use a new tip for each solution.
2. Add 200 μl of prepared wash solution 1 to the 96 deep well plates marked FW1 and SW1.
3. Add 300 μl of prepared wash solution 2 of the 96 deep well plates marked FW2 and SW2.
4. Add 60 μl of nuclease free water to wells of the 96-standard plate marked Elution.
5. Place a 96 tip comb in a 96-standard plate.
55m
Preparing sample plate and running the extraction program
7. Aliquot 400 μl of the Lysis/Binding solution supplemented with carrier RNA and 100% Isopropanol (see step 9 and 10 in Reagent preparation) to a 96 deep-well plate marked as Sample plate.
8. Add the recovered volume (200-240 μl) of samples to the 96 deep-well plate containing the Lysis/Binding Solution. Mix by gently pipetting up and down a few times. (Discard tube containing the punches).
Note
When adding sample, check and confirm the sample ID. Immerse pipette tips slightly in the lysis/binding solution to prevent creating aerosols and rinse pipette tip. Keep all other tubes closed to avoid cross contamination.
9. Add 14 μl of prepared bead mix to the wells of the Sample plate containing the Lysed sample solution using a new tip for every addition and rinsing it with the sample solution to prevent loss of beads.
10. Check to confirm that the instrument is set up with 96 deep-well magnetic head and 96 deep-well heat block. Select the MVRI_Flex_SV_300ul protocol on the equipment and load the plates onto the King Fisher Flex as directed, then start the protocol.
11. Close the front lid of the device.
30m
Un-loading the device:
12. After completion of the run, a final prompt will appear. “Unload plate containing the RNA”.
13. Transfer eluted viral RNA to labelled containers and keep on ice for immediate use or store frozen until needed.
14. Empty and wipe clean equipment. Dispose of processing plates and their contents by following standard laboratory processes.
5m