Table 1.

The original QIAamp MinElute Virus Spin Kit protocol and its optimized versions

Step*	Original protocol	Optimization A	Optimization B	Optimization C
N/A	No sample pretreatment	†	†	Add 70 µL of Urine Conditioning Buffer to 1 mL of sample, vortex, centrifuge at 3,000 × g for 15 minutes, discard supernatant
1	Pipet 25 μL of Protease into a tube	WB only—50 μL of Protease	50 μL of Protease	Resuspended pellet in a mix of 200 µL AVE, 200 µL AL/carrier RNA, and 25 µL Protease
2	Add 200 μL of sample	WB only—200 µL of packed blood cells and 200 µL of PBS	†	N/A
3	Add 200 μL of buffer AL/carrier RNA, vortex	WB only—400 μL of buffer AL	†	N/A
4	Incubate at 56°C for 15 minutes	†	30 minutes	†
N/A	N/A	†	Add 1.6 mL of buffer RLT Plus/1% βME, incubate at RT for 5 minutes	†
5	Briefly centrifuge the tube to remove drops from the inside of the lid	†	†	†
6	Add 250 μL of 100% ethanol, vortex. Incubate for 5 minutes at room temperature	WB only—500 µL of ethanol	1.4 mL of ethanol	†
7	Briefly centrifuge the tube to remove drops from the inside of the lid	†	†	†
8	Apply onto the column. Centrifuge at 21,100 × g for 1 minute. Place the column in a clean collection tube	Centrifuge at 900 × g for 1 minute, followed by 1 minute 21,100 × g	Apply onto the RNeasy Mini Spin Column. Centrifuge at 900 × g for 1 minute, followed by 1 minute 21,100 × g	†
9	Add 500 μL of buffer AW1 without wetting the rim. Centrifuge at 21,100 × g for 1 minute. Place the column in a clean collection tube	WB only – repeat step 9	†	†
10	Add 500 μL of buffer AW2 without wetting the rim. Centrifuge at 21,100 × g for 1 minute. Place the column in a clean collection tube	†	†	†
11	Add 500 μL of 100% ethanol without wetting the rim. Centrifuge at 21,100 × g for 1 minute. Place the column in a clean collection tube	†	†	†
12	Place the column in a clean collection tube. Centrifuge at 21,100 × g for 3 minutes	†	†	†
13	Place the column into a new collection tube, open the lid, and incubate the assembly at 56°C for 3 minutes	†	†	†
14	Place the column in a clean tube. Apply 30 of buffer AVE to the center of the membrane. Close the lid and incubate at room temperature for 1 minute. Centrifuge at 21,100 × g for 1 minute	Incubate at 56°C for 5 minutes	Incubate at 56°C for 5 minutes, centrifuge at 21,100 × g for 1 minute, reload eluate, incubate at 56°C for 1 minute, centrifuge at 21,100 × g for 1 minute	†

βME = β-mercaptoethanol; WB = whole blood. The modifications to the original protocol are indicated per optimization versions.

* Step number is according to the manufacturer’s original protocol.

† No modification made to the original protocol.