L methyl 3h methionine

We reverse transfected 75,000 HeLa cells with the appropriate siRNA as described under siRNA inactivation and total RNA extraction. After a 72-h incubation, the cells were washed and incubated for 30 min at 37°C in methionine-free DMEM (Invitrogen) supplemented with 10% dialyzed FBS (Sigma-Aldrich), labeled for 30 min with 50 μCi l-(methyl-³H)-methionine (50 mCi/ml; PerkinElmer), washed with complete growth medium supplemented with 0.3 mg/ml methionine, and incubated for 0, 15, 30, 60, 120, or 240 min in the same medium. The cells were then resuspended in 1 ml TRI Reagent (Life Technologies) for RNA extraction as described. Purified RNAs were separated on an agarose denaturing gel (6% formaldehyde/1.2% agarose in HEPES-EDTA buffer) for 16 h at 60 V and transferred to a GeneScreen membrane. The membrane was sprayed with tritium enhancer (PerkinElmer) and exposed and autoradiographed.

Cells were grown on a 60-mm plate. Cells were starved for 15 in pre-warmed methionine-free DMEM with dialyzed FBS at 37°C in a CO₂ humidified cell culture incubator. l-Methyl-[³H]-methionine (Perkin-Elmer) was added to a final concentration of 50 μCi/ml. RNA was labeled for 30 min before replacing media with pre-warmed complete DMEM with 10% FBS supplemented with 0.3 mg/ml methionine. Cells were collected at indicated times and RNA was extracted using Trizol. Five micrograms of extracted RNA were resuspended in RNA loading dye and heated to 85°C for 10 min before placing on ice. Denatured RNA was electrophoresed as for [³²P]-metabolic labeling. The following day, the gel was subjected to mild alkaline treatment (10 min in 50 mM NaOH/10 mM NaCl), neutralization (10 min in 2.5× TBE) and equilibration in 2× SSC while rocking. RNA was transferred overnight by passive transfer to Hybond N⁺ Nylon membrane using 20× SSC. The following day, nucleic acids were immobilized on the membrane by UV crosslinking. Membranes were exposed to Amersham Tritium phosphor screen for at least five days before detection using Typhoon phosphorimager.

Pulse chase labeling of rRNA was performed essentially as described [54,55]. Briefly, 20 ml cultures of yWO3 containing pWO58 or pWO116 were grown at 30°C in SC-Met-Leu media containing 2% dextrose to an OD₆₀₀ of 0.4. 10 ml of cells were harvested then resuspended in 3 ml SC-Met-Leu media containing 2% dextrose. Cells were allowed to grow at 30°C for 25 min. 250μCi of L-[Methyl-³H]-Methionine (Perkin-Elmer) was added and cells were allowed to grow two minutes. L-Methionine was then added to a final concentration of 0.6 mM. A 600μl cell aliquot was immediately collected, spun down for 15 sec at 3,000 rpm, supernatant removed, and placed in a dry ice/methanol bath. Time points were collected in the same manner at 1, 2, 4, and 8 minutes after methionine chase. Total RNA was extracted via hot phenol method as previously described. 20,000 cpm from each time point was loaded on a 1.25% agarose gel (containing formaldehyde and MOPS) and electrophoresed for 8 hours at 70V. The RNA was then transferred to a nylon membrane, UV crosslinked, and the membrane allowed to dry. The membrane was then coated four times with ENH³ANCE (Perkin-Elmer), allowing 15 minutes for drying in between each coat and after the final coat, as per manufacturer’s instructions. Autoradiography film was exposed to the membrane for 96 hr at −80°C prior to development.