Materials and Reagents

W303a cell line
Sodium acetate trihydrate (CH₃CO₂Na·3H₂O) (Sigma-Aldrich, catalog number: 236500 )
Phenol (C₆H₅OH) (Sigma-Aldrich, catalog number: P1037 )
EDTA (Na₂EDTA·2H₂O) (Sigma-Aldrich, catalog number: ED2SS )
Acetic acid (CH₃COOH) (Sigma-Aldrich, catalog number: 320099 )
Chloroform (CHCl₃) (Sigma-Aldrich, catalog number: 472476 )
8-hydroxyquinoline (C₉H₇NO) (Sigma-Aldrich, catalog number: 252565 )
NaCl (Thermo Fisher Scientific, catalog number: S641-500 )
Synthetic complete (SC) medium
SDS (Sigma-Aldrich, catalog number: L3771 )
Isoamyl alcohol (Sigma-Aldrich, catalog number: W205710 )
Ethanol (Thermo Fisher Scientific, catalog number: 64-17-5 )
DEPC water
Phenol-AE buffer (see Recipes)
Phenol: CHCl₃/AE-Na (see Recipes)
CHCl₃: Isoamyl alcohol (24:1) (see Recipes)

Equipment

Standard bench-top centrifuge
Microfuge
Shaker
1.5 eppendorf tube
Liquid nitrogen

Procedure

Prepare reagents according to recipes. Note that everything is in DEPC water.
Inoculate W303a cells expressing different TOR1-RR variants in 2 ml SC medium overnight.
Subculture the cells starting from OD₆₀₀=0.1 in 10 ml SC media, shake vigorously at 30 °C, 300 rpm for around 4-6 h until OD₆₀₀=0.4-0.5.
Collect the cells by spinning down without freezing on ice. Discard supernatant.
Re-suspend cells with 1 ml water and transfer to a 1.5 eppendorf tube, quickly spin down at 3,000 x g for 15 sec.
Re-suspend cell pellet in 400 µl of AE buffer at room temperature.
Add 40 µL 10% SDS (final around 1%) and vortex briefly at room temperature (RT).
Immediately add 500 µl hot phenol/AE (put in 65 °C for 10 min before use), vortex vigorously for 1 min.
Incubate at 65 °C for 5 min. Briefly vortex every 30 sec.
Immediately freeze by dumping into liquid nitrogen (labels won’t get off).
Wait to thaw at RT (put in 30 °C to thaw may crack the tube).
Centrifuge for 10 min on a standard laboratory microfuge at 20,000 x g at RT.
Transfer around 400 μl supernatant to a new eppendorf tube. Recycle the lower phenol fraction carefully following the chemical safety protocol in your laboratory.
Add equal volume (400 μl) phenol: CHCl₃/AE-Na. Vortex vigorously for 1 min at RT.
Spin down at 20,000 x g for 5 min in a standard laboratory microfuge.
Transfer supernatant (around 350 μl) to a fresh 1.5 ml eppendorf tube.
Add CHCl₃: isoamyl alcohol (24:1). Vortex vigorously for 1 min at RT.
Transfer aqueous supernatant to fresh 1.5 ml microfuge tube. If white cloudy precipitate is observed between aqueous phase and organic phase, repeat steps 17-18.
Add 1/10 volume of 3 M NaOAc (pH 5) and vortex vigorously. Add 2.5 volumes of ethanol. Vortex again.
Place at -20 °C for at least 30 min.
Spin down in the microfuge at 20,000 x g, 15 min at 4 °C. RNA pellet is usually visible.
Add ice-cold 75% EtOH, place at 4 °C for around 10 min. Vortex and spin down on microfuge 20,000 x g, 15 min at 4 °C.
Discard supernatant. Suck out the liquid droplets in the tube.
The white RNA pellet will turns clear when it dries out. Add 30-50 µl ddH₂O (DEPC) immediately after it becomes clear.
Do not let the RNA over-dry, which will make it difficult to dissolve. If RNA pellet is over-dry, dissolve RNA at 37 °C for 30 min.
Store RNAs at -80 °C for more than 2 months.

Recipes

Phenol-AE buffer
Make Acetate-EDTA(AE) buffer
50 mM NaOAc
10 mM EDTA (pH 5.0)
Liquefied phenol equilibrated with equal volume of AE buffer. Store at 4 °C.
Phenol: CHCl₃/AE-Na
Make AE-Na buffer
10 mM NaOAc
2 mM EDTA
100 mM NaCl (pH 6.0)
50% phenol/AE
50% CHCl₃
0.25% 8-hydroxyquinoline
Store at 4 °C.
CHCl₃: Isoamyl alcohol (24:1)

Acknowledgments

This protocol was adapted from and used in Wei and Zheng (2009) and Wei et al. (2009).

References

Wei, Y. and Zheng, X. F. (2009). Sch9 partially mediates TORC1 signaling to control ribosomal RNA synthesis. Cell Cycle 8(24): 4085-4090.
Wei, Y., Tsang, C. K. and Zheng, X. F. (2009). Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1. EMBO J 28(15): 2220-2230.