TITLE: Purification of viral RNA from cell culture isolates or FTA cards using MagMAX Viral RNA Isolation Kit
AUTHORS: Erika Bujaki, Dimitra Klapsa, Kafayat Arowolo, Joyce Akello, Nick Grassly, Javier Martin

[DESCRIPTION]
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. 

[STEPS]
SECTION: Reagent Preparation
1.1. 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.
SECTION: Reagent Preparation
1. 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.
SECTION: RNA extraction
5. Automated extraction using King Fisher Flex – Workflow C

Read the King Fisher Flex instrument manual for installation and operating instructions in its entirety before operating the magnetic particle processor. 

Continued from step 2.

Plasticware for small volume sample
extractions on King Fisher Flex equipped with 96-tip magnetic head.
 
 
 
SECTION: Reagent Preparation
1.2. 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.


SECTION: FTA card Processing
2. 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.

SECTION: Reagent Preparation
1.3. 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.
SECTION: Reagent Preparation
1.4. 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.


 

SECTION: Reagent Preparation
1.5. 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
 
 
Add 100% isopropanol and mix well by vortexing.  
 
               

 

SECTION: Reagent Preparation
1.6. Bead Mix
Vortex the nucleic acid binding beads well to ensure that the beads are fully resuspended.
Combine the components that are listed below:
 
 
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.


 

SECTION: RNA extraction
3. Manual extraction – Workflow A

The indicated processing times are based on handling 12 samples at the same time .
SECTION: RNA extraction
3.1. 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). 

 
3. Mix gently by vortexing for 30s and spin briefly to collect tube content.
SECTION: RNA extraction
3.2. Bead capture and washes:

4.  Add 14 μl of prepared bead mix to each sample tube containing the lysed sample solution.

 
5.  Mix tubes thoroughly at gentle speed for 4 min to fully lyse viruses and bind RNA to beads. 

 
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.

 
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.

 

 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.

 
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. 

 

SECTION: RNA extraction
4. Automated extraction using King Fisher Duo Prime – Workflow B

Read the King Fisher Duo Prime instrument manual for installation and operating instructions in its entirety before operating the magnetic particle processor. 

Continued from Step 2.

Plasticware for small volume sample extractions on King Fisher Duo Prime equipped with 12-tip magnetic head.
 
 
SECTION: RNA extraction
3.3. 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. 

 
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.


 

 

 

SECTION: RNA extraction
4.1. Preparing 96 deep well plate:

 

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.  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).

 
3.  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.

 
4.   Set up plate by aliquoting the required reagents into the appropriate positions as shown in table below:

5.   Add 200 μl of prepared wash solution 1 to rows B and C of the 96 deep well plate.

6.   Add 300 μl of prepared wash solution 2 to rows D and E of the 96 deep well plate.

7.   Place a 12-tip comb in a 96 deep well plate in Row H.

8.  Add 60 μl of Elution buffer/ nuclease free water to the elution strip tube.
SECTION: RNA extraction
4.2. Setting up and running the King Fisher Duo Prime:

9.  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.

10.   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.

11.   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.

12.  Select the MVRI_DUO_SV_300ul Protocol and press start.

13.  Close the front lid while the KingFisher is running.
SECTION: RNA extraction
4.3. Un-loading the device:

14.   After completion of the run, a final prompt will appear.  “Unload RNA plate and RNA Elution Strip”.

15.   Unload the elution strip containing the RNA, cap and place the elution strip containing the RNA on ice. 

16.   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.

17.   Transfer eluted viral RNA to  labelled containers and keep on ice for immediate use or store frozen until needed.
SECTION: RNA extraction
5.1. Preparing sample plate

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. 

2.  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).


 

SECTION: RNA extraction
5.2. Setting up the processing plates and running the extraction program 

3.  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.

4.  Label and prepare the processing plates according to the table below:

 

 
 

 5.  Add 200 μl of prepared wash solution 1 to the 96 deep well plates marked FW1 and SW1.

 6.  Add 300 μl of prepared wash solution 2 of the 96 deep well plates marked FW2 and SW2.
 
7.  Add 60 μl of nuclease free water to wells of the 96-standard plate marked Elution.
 
8. Place a 96 tip comb in a 96-standard plate. 
 
9. 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.

10.  Close the front lid of the device.

SECTION: RNA extraction
5.3. Un-loading the device:

11.  After completion of the run, a final prompt will appear. “Unload plate containing the RNA”.

12. Transfer eluted viral RNA to  labelled containers and keep on ice for immediate use or store frozen until needed.

13.  Empty and wipe clean equipment. Dispose of processing plates and their contents by following standard laboratory processes.
 
SECTION: FTA card Processing
2.1.  Set the thermal block to 70°C.
SECTION: FTA card Processing
2.2. For each sample:
Set up and label clean nuclease free 1.5 mL microcentrifuge tubes for each FTA card to be processed.

SECTION: FTA card Processing
2.3. 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.
SECTION: FTA card Processing
2.4. 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).


 

 

SECTION: FTA card Processing
2.5. Dispose of the biopsy punch in the sharps bin and use new punch for each card.


SECTION: FTA card Processing
2.6. 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).

SECTION: FTA card Processing
2.7. Aliquot 300 μl of RNA Processing Buffer into each tube with the FTA card discs.
SECTION: FTA card Processing
2.8. Close and vortex tubes at high speed for 40 seconds.

SECTION: FTA card Processing
2.9. 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.)


 

SECTION: FTA card Processing
2.10. When incubation is over, 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.