[Bradley Stevenson]

My colleagues have been successful in recovering viral RNA from the transport media using the following protocol and Qiagen RNEasy columns. We are starting there and trying to simply replace the filter cartridge with steps using carboxylated GE Healthcare Speedbeads. We are assuming that binding will be same (unknown) in the lysis and binding buffers in the attached protocol. In other words, I dont think you will need to concentrate your sample.  We are literally sitting down to work on this now with things I moved from my lab. I will let you know how it goes.

Hillbilly Bead Viral RNA Extraction

Viral Lysis Buffer (AVL) – In House – 50 mL Recipe

• Guanidine Thiocyanate – 35.44 g
• Tris Base – 0.61 g
• RNAase Free Water – Bring to Volume 50 mL
• pH 7.3-7.7

 

Carrier RNA

• 1 mL AVL contains 10 ug Carrier RNA
• 1 mammalian cell contains approx. 20 pg RNA
• Add 5 x 10⁵ hybridoma cells per mL AVL

 

Hillbilly Bead Preparation (a.k.a Preparation SpeedBead Magnetic Carboxylate Modified Particles)

• SpeedBead Magnetic Carboxylate Modified Particles (GE Healthcare; 65152105050250)
• NaCl (e.g. Sigma Aldrich,S3014-500G)
• 1 M Tris-HCl, pH 8.0
• 5 M EDTA, pH 8.0
• DEPC-treated water

 

For 50mL of Hillbilly Beads

1. To a sterile 50 mL conical tube, add 2.92 g NaCl with ~40 mL of water, vortex to dissolve. Add 0.5 mL of 1M Tris-HCl and 0.1 mL of 0.5 M EDTA and add water to 50 mL. This is the TE to be used next.
2. Thoroughly resusped particles in stock of SpeedBead Magnetic Carboxylate Modified Particles. Transfer 1 mL to a 1.5 mL microcentrifuge tube.
3. Place beads in rack on magnet and wait until solution is cleared (2-3 min). Carefully remove liquid with pipettor, do not aspirate beads.
4. Add 1 mL of TE (freshly prepared above) and resuspend particles. Place on magnetic rack and allow solution to clear. Carefully remove liquid.
5. Repeat washing beads with TE two more times.
6. Resuspend beads in 1 mL TE and set aside.
7. To a new sterile 50 mL conical tube, add 2.92 g NaCl with ~40 mL of water, vortex to dissolve. Add 0.5 mL of 1M Tris-HCl and 0.1 mL of 0.5 M EDTA and add water to 49 mL.
8. Transfer TE-washed particles to prepared solution and mix. This is the Working Stock of particles (2% SpeedBead Magnetic Carboxylate Modified Particles, 1M NaCl, 10 mM Tris-HCl, 1 mM EDTA).
9. Store at 4oC for up to 3 months. Mix well before each use.

 

Wash Buffer 1 (AW1) – In House

• 6M Guanidine HCl – 19 mL
• 95% EtOH – 25 mL
• pH 4.5-5.5

 

Wash Buffer 2 (AW1) – Use Promega RNA Wash Solution

 

Elution Buffer (AVE)

• RNAase Free water

 

Procedure

1. Pipet 560 uL prepared Buffer AVL containing carrier RNA into a 1.5 mL microcentrifuge tube.
2. Add 140 uL of sample (in Viral Transportation Medium). Mix by pulse-vortexing for 15 s.
3. Incubate at Room Temperature (15-25oC) for 10 min.
4. Briefly centrifuge to remove any liquid from inside cap.
5. Add 560 uL of 95-100% Ethanol. Mix by pulse-vortexing for 15 s.
6. Add 40 uL of Bead Solution. Mix by vortex for 5 min.
7. Place tube on magnet to pellet beads (2-5 min or until solution is cleared).
8. Remove buffer, being careful not to aspirate beads
9. Remove tube from rack, add 560 uL of Buffer AW1. Mix by pulse-vortexing for 15 s.
10. Place tube on magnet to pellet beads.
11. Remove buffer, being careful not to aspirate beads.
12. Remove tube from rack, add 500 uL of Buffer AW2, mix by pulse vortexing.
13. Place tube on magnet to pellet beads.
14. Remove buffer and repeat wash with AW2.
15. Dry beads at 50oC for 15 min to remove traces of ethanol.
16. Add 60 uL Buffer AVE. Mix by pulse-vortexing for 15 s. Incubate for 5 min. at room temperature.
17. Place tube on magnet to pellet beads.
18. Transfer RNA-containing solution to a clean tube.