TITLE: Large volume viral RNA extraction using MagMAX Viral RNA Isolation Kit
AUTHORS: Erika Bujaki, Thomas Wilton, Dimitra Klapsa, Alison Tedcastle, Joyce Akello, Nick Grassly, Javier Martin

[DESCRIPTION]
This method describes large volume nucleic acid purification from sewage concentrates starting with 1.2 mL of sample using MagMAX ‱ Viral RNA Isolation Kit. To perform automated extraction, follow steps in Workflow A, and for manual extractions follow Workflow B diverging after the initial sample processing with Lysis/Binding solution. Appendix 1 contains pictures providing a visual guide to manual magnetic beads based nucleic acid extraction.

[STEPS]
2. Sample Processing
3.                                    Workflow  A  - Automated extraction                                
1.1. 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.
1. Reagent
preparation
1.2. 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.

 

2.1. Prepare the lysate in 5mL or 15mL centrifuge tubes

For each sample: 
Set up and label 5ml Eppendorf tubes, then aliquot 2.4 ml of the lysis/binding solution (supplemented with carrier RNA and 100% Isopropanol - see step 9 and 10 in Reagent preparation) into each tube.
Add 1.2 ml of the samples to the tubes containing the Lysis/Binding solution. 
·      
 

 
Mix gently by vortexing for 30s and spin
     briefly to collect tube content.
1.3. Lysis/Binding Solution

Combine the components listed below in the order indicated.
Prepare enough solution for the number of samples extracted that day, including controls, with allowing extra 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.  

 

1.4. Bead Mix

Combine the components that are listed below. Prepare enough mixture for the number of samples extracted that day with allowing extra for pipetting loss.

 


 Vortex the nucleic acid binding beads well to ensure they are fully resuspended.
 
 
 
Mix well by vortexing and store appropriately until needed.


3.1. Setting up and running  the 24 deep well plates

   Label two 24 deep-well plates as Plate 1 and Plate 2.
   Transfer the 3.6 ml of sample/lysis mixture to row A of plate 1.
   Add 60 μl of prepared Bead Mix to wells in row A of the 24 deep well plate 1 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. Centrifuge tubes briefly to collect content.

 
Aliquot the required reagents into the plates according to the table above:

5.    Add 900 μl of prepared Wash Solution 1 to rows B and C of Plate 1.
6.    Add 1350 μl of prepared Wash Solution 2 to rows A and B of Plate 2.
7.    Place a 12-tip comb in a 96 deep well plate in row D of Plate 1.
8.   Add 50 μl of Elution Buffer or nuclease free water to row D in Plate 2.
9.   Check to confirm that the King Fisher Duo Prime is set up with 6 tip magnet and heating block.
10.  Select the program MVRI Duo LV 1200ul and load the two plates using the A1 markings as prompted. The two plates will be on opposing sides of the turning platform in the processor.
11.   Start the program and close the front lid while the King Fisher is running.
12.   After completion of the run, a final prompt will appear to unload the plates.
13.   Press the “Check Mark”, then unload the plate containing the eluted viral RNA. Transfer the RNA
to labelled containers and store appropriately until further use.

 

14.  Unload the sample plate and empty contents into beaker with disinfectant. Switch on UV light for
disinfection by selecting the program in the maintenance protocols.

Changing the magnetic head on the Kingfisher Duo Prime

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 12/6 tip protocol with a dummy test plate containing the tip comb in the required row to ensure the right positioning. Unload the test plate.
4. Workflow B - Manual extraction

Continued from Step 2.
4.1. Bead capture and washes:

  Add 60 μl of prepared bead mix to each sample tube containing the lysed sample solution using a new tip for every addition and rinsing it with the sample solution to prevent loss of beads. 

 

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

 

3.    Place processing tubes on magnet and leave for at least 3 minutes to allow for bead capture to complete when beads form a pellet against the magnet.

 

 4.    Carefully aspirate and discard supernatant without disturbing the beads. 
5.     It is important to remove the lysis supernatant fully, so a brief centrifugation before collecting the remaining supernatant might be necessary.
6.     Remove tube from magnetic stand and place in tube rack for washing with Wash Solution 1.
7.     Add 300 µl Wash Solution 1, supplemented with Isopropanol to each sample and vortex at moderate speed for 30s.

 

8.     Centrifuge briefly to collect tube content.
9.     Capture beads on magnet for 3-5 min or until mixture becomes clear, indicating full capture.
10.   Carefully aspirate and discard supernatant.
11.    Repeat steps 7-10 one more time to complete two washes with Wash Solution 1.
12.   Remove tube from magnetic stand and place in tube rack for washing with Wash Solution 2.
13.   Add 450 µl Wash Solution 2 supplemented with ethanol to each sample and vortex at moderate speed for 30s.

 
14.   Centrifuge briefly to collect tube content.
15.   Capture beads on magnet for 3-5 min or until mixture becomes clear, indicating full capture.
16.   Carefully aspirate and discard supernatant. 
17.   Repeat steps 13-16 to complete two washes with Wash Solution 2. It is important to completely remove the supernatant after the second wash to avoid inhibition in downstream applications.
4.2. Drying the beads and elution:

18.   Centrifuge briefly and remove any residual solution with
fine-tipped pipette.
19.   Dry the beads by leaving the tube open for 2 minutes to
20.   Add 50 µL Elution Buffer to each sample and shake/vortex vigorously for 4 min.allow any remaining alcohol to evaporate.

 


21.   Centrifuge briefly to collect tube content.
22.   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.