vRNA extraction

For sequencing runs 1-3, we extracted viral RNA from buccal swabs manually, using the protocol described below. For sequencing run 4, we had access to a Roche Magnapure RNA extraction robot, which we used. Both methods produced good quality RNA. For the manual extraction, we have provided the protocol below.

This protocol follows the published protocol for vRNA extraction with the QiAmp viral RNA mini kit (Qiagen, catalogue # 52906). Most mumps samples are collected as buccal (cheek) swabs, which are likely to contain host cells and proteins. To remove host cells and maximize the amount of material that we could extract, we added 2 additional spin steps to the beginning of the standard protocol. The first spin pellets out and removes host cells. The second spin concentrates virions at the bottom of the tube. Excess supernatant is removed and the virions are resuspended in the ~140-200 ul necessary for the start of the protocol, concentrating the sample. All parts of this protocol should be carried out in a clean, pre-PCR hood that has been wiped down with bleach and ethanol before use. Until particle lysis is complete (step 5), there are infectious virions in the sample. All pipette tips and tubes should be discarded and soaked in 10% bleach for 1 hour to sterilize.

Concentrate vRNA and remove host cells

1. Thaw 500 µl of buccal swab. Transfer to a 1.5 ml Eppendorf tube.
2. Centrifuge at 5,000 x g for 5 minutes at 4°C to pellet host cells. After spin is complete, transfer supernatant to a clean tube and discard tube with pellet.
3. Centrifuge supernatant for 90 minutes at 4°C at maximum speed (14,000 rpm) to concentrate virions in the lower portion of the supernatant.

** About 20 minutes before the spin completes, prepare the buffer AVL with carrier RNA. For each sample you are extracting, you will need 0.56 ml of buffer AVL + 5.6 µl of carrier RNA suspended in buffer AVE. The first time you suspend carrier RNA in buffer AVE, aliquot into tubes and store at -20°C to avoid multiple freeze-thaws.

1. Remove ~300 µl of liquid from the top of the supernatant to leave 140-200 µl of total supernatant. Pipet up and down to resuspend viral pellet.

QiAmp viral RNA mini kit protocol

This protocol follows directly from the manufacturer’s recommended protocol.

1. Add 560 μl of of the Buffer AVL/carrier RNA mixture into the 1.5 ml microcentrifuge tube containing your resuspended virions. Vortex for 15 seconds, then spin down to remove droplets from the lid. It is critical to thoroughly mix your sample to ensure efficient lysis.

2. Incubate at room temperature for 10 min. After 10 minutes, lysis is complete and the sample is no longer infectious.

3. Add 560 μl of ethanol (96–100%) to the sample, mix by pulse-vortexing for 15 seconds, and spin down.

4. Transfer 630 μl of the solution from step 5 to the QIAamp Mini column (in a 2 ml collection tube) without wetting the rim. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini column into a clean 2 ml collection tube, and discard the tube containing the filtrate.

5. Repeat step 8 until all sample has been run through the filter column.

6. Add 500 μl of Buffer AW1. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini column in a clean 2 ml collection tube (provided), and discard the tube containing the filtrate.

7. Carefully open the QIAamp Mini column, and add 500 μl of Buffer AW2. Close the cap and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min.

8. Place the QIAamp Mini column in a new 2 ml collection tube, and discard the old collection tube with the filtrate. Centrifuge at full speed for 1 min.

9. Place the QIAamp Mini column in a clean 1.5 ml microcentrifuge tube. Discard the old collection tube containing the filtrate. Carefully open the QIAamp Mini column and add 30 μl of Buffer AVE equilibrated to room temperature. Close the cap, and incubate at room temperature for 1 min. Centrifuge at 6000 x g (8000 rpm) for 1 min.

** Store eluted viral RNA at -80°C.