TRI Reagent® Sigma

Protocol tips

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- 1-Bromo-3-chloropropane is less toxic than chloroform and its use for phase separation decreases the possibility of contaminating RNA with DNA. - The chloroform used for phase separation should not contain isoamyl alcohol or other additives.

Upstream tips
- 1-Bromo-3-chloropropane is less toxic than chloroform and its use for phase separation decreases the possibility of contaminating RNA with DNA. - The chloroform used for phase separation should not contain isoamyl alcohol or other additives.

Publication protocol

Cells of two culture replicates for each cell line were harvested at two different time points during mid-exponential phase. Adherent cells were washed with DPBS, no calcium, no magnesium (PAA) and detached using trypsin (Life Technologies, Carlsbad, CA). RNA was isolated using TRI reagent® (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s protocol. In brief, up to 5 × 106 cells were homogenized in 500 μl TRI reagent® and incubated for 5 min at room temperature. The phases were separated by the addition of 0.1 ml chloroform and centrifugation. Isopropanol was added to the aqueous phase to precipitate the RNA. After centrifugation, the pellets were washed with 75% ethanol and air-dried. The pellets were then dissolved in 30 μl nuclease free water and the quality and quantity were determined based on the absorption at 260 nm, A260/280 and A260/230 ratio in a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA). The RNA integrity number (RIN) was evaluated by capillary electrophoresis using RNA Series II 6000 W. Nano Kit (Agilent Technologies Inc., Santa Clara, CA) and a 2100 Bioanalyzer (Agilent). Only samples with an A260/230 and an A260/280 of 2 ± 0.1 and a RIN > 7 were used.

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Manufacturer protocol

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