Isolation and Visualization of Rotavirus Genome via Gel Electrophoresis

Resumo

Source: Philip, A. A., et al., Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins. J. Vis. Exp. (2020)

This video demonstrates the isolation of rotavirus double-stranded RNA from clarified cell lysates. It further illustrates the separation and visualization of genome segments through polyacrylamide gel electrophoresis.

Protocolo

Gel electrophoresis of viral dsRNA

1. Place 600 µL of clarified infected cell lysates and 400 µL of guanidinium thiocyanate in 1.5 mL microcentrifuge tubes, vortex for 30 s, and incubate at room temperature for 5 min. Add 200 µL of chloroform, vortex for 30 s, and incubate for 3 min. After centrifuging for 5 min at 13,000 x g (4 °C), transfer ~550 µL of the upper aqueous phase into fresh tubes.
2. Recover viral dsRNA from the aqueous phase by adding 2 volumes (~900 µL) of cold isopropyl alcohol and inverting tubes 4−6x. After incubating at room temperature for 10 min, centrifuge tubes for 10 min at 13,000 x g (4 °C). Discard supernatants and retain the RNA pellets.
3. Wash the pellets by adding 1 mL of 75% ethanol to the tube, inverting once, and centrifuging for 5 min at 7,500 x g (4 °C). After carefully removing the ethanol wash with a pipettor, allow RNA pellets to air-dry on the lab bench for 5−10 min. Dissolve RNA pellets in 15 µL of nuclease-free molecular biology grade water and store at -20 °C.
4. Combine 10 µL of dissolved RNA samples with 2 µL of 6x DNA loading buffer, load onto precast 10% polyacrylamide mini gels (or hand-cast equivalents), and resolve RNAs by electrophoresis in Tris-glycine running buffer for 2 h under a constant current (16 mA). Soak gels for 5−10 min in water containing ~1 µg/mL ethidium bromide and detect rotavirus genome segments with a UV transilluminator.

Materiais

Lista de materiais utilizados neste artigo

Nome	Empresa	Número de catálogo	Comentários
Bio-Rad 8-16% Tris-Glycine Polyacrylamide Mini-Gel	Bio-Rad	45608105
Cellometer AutoT4 viable cell counter	Nexcelom
ChemiDoc MP Gel Imaging System	Bio-Rad
Chloroform	MP	194002
Complete Protease Inhibitor	Pierce	A32965
Disposable Transfer Pipettes, Ultrafine Extended Tips	MTC Bio	P4113-11
Ethanol, Absolute (200 proof)	Fisher Bioreagents	BP2818-500
Ethidium Bromide Solution (10 mg/ml)	Invitrogen	15585-011
Isopropyl alcohol	Macron	3032-02
NanoDrop One Spectrophotometer	ThermoScientific
Nuclease-Free Molecular Biology Grade Water	Invitrogen	10977-015
Tris-Glycine-SDS Gel Running Buffer (10x)	Bio-Rad	161-0772
Triton X 100	Fisher Bioreagents	BP151-500
Trizol RNA Extraction Reagent	Ambion	15596026