3.1.1. Inactivation of Endogenous Ribonucleases
1. Prepare ribonuclease-free water by adjusting the volume of water required to contain 0.2% (w/v) DEPC.
2. Incubate the treated water at 37°C for 12 h and then autoclave at 121°C for 30 min.
3. Immerse glassware and nonsterile plasticware in a solution of 0.2% (v/v) DEPC and then autoclave at 121°C for 30 min.
4. Prepare buffers used in RNA procedures using DEPC-treated water. Buffers that cannot be autoclaved should be sterilized by filtration through a 0.2-^m filter.
1. Into a sterile 1.5-mL Eppendorf tube add the required volume of fecal filtrate (typically 400 ^L). Adjust the volume to contain 1% (w/v) SDS and 100 ^g/mL of proteinase K. Mix by pipeting and incubate at 37°C for 1 h.
2. To each reaction mixture add an equal volume of phenol/chloroform mixture (1:1) and vortex the sample for 30 s.
3. Centrifuge at 11,000g for 10 min, which separates the sample into two phases.
4. Collect the aqueous phase into a new sterile 1.5-mL Eppendorf tube and add an equal volume of chloroform.
5. Mix the sample by vortexing for 30 s followed by centrifugation at 11,000g for 10 min. Again collect the aqueous phase.
6. To precipitate the template 11 dsRNA segments, add 2 vol of cold absolute ethanol to the aqueous phase, and incubate the sample at -80°C overnight (12-18 h).
7. Recover the purified dsRNA by centrifugation (11,000g for 10 min) and then resuspend the pellet of RNA obtained in 100 ^L nuclease-free water.
1. Incubate the total RNA recovered (approx 100 ^L) with 2 ^L DNase 1 and 10.2 ^L 10X Tris buffer (to give a final working solution of 1X) at 37°C for 30 min. This step allows sufficient time for any copurifying chromosomal DNA to be degraded.
2. To this solution add 80 ^L of phenol/chloroform (3:1) reagent and vortex the sample for 1 min.
3. Centrifuge at 11,000g for 5 min and collect the aqueous phase as before into a sterile 1.5-mL Eppendorf tube.
4. Finally, precipitate the DNA-free purified dsRNA with 10 ^L of 3 M sodium acetate and 300 ^L of cold absolute ethanol.
5. Incubate the above overnight at -20°C and recover the dsRNA by centrifugation for 10 min at 10,000 rpm.
6. Wash the dsRNA pellet with 1 mL of cold, 70% (v/v) ethanol and resuspend the pellet in 100 ^L DEPC-water.
7. Purified RNA can be analyzed by conventional agarose gel (1.5%) electrophoresis and rotaviral genomic segments visualized after staining with ethidium bromide (0.1 mg/mL). Store the dsRNA at -80°C.
Was this article helpful?