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

otal RNA was isolated from 5 × 106 cells/ml using TRI Reagent (Sigma-Aldrich) for DMSO-treated and CHX-treated EL4 T cells, unstimulated (0 h) or stimulated for 4 h with PMA/I. Briefly, cells were pelleted at 1500 rpm (Beckman Allegra 6R Centrifuge) for 5 min at room temperature, resuspended in 1 ml of TRI Reagent and incubated at room temperature for at least 10 min to allow complete dissociation of nucleoprotein complexes. 200 μL of chloroform was added and samples were vortexed vigorously and incubated on ice for 15 min. The samples were then centrifuged at 13 000 rpm (Eppendorf Centrifuge 5415 R) for 15 min at 4 °C, after which the aqueous phase was transferred to a new 1.5 mL tube and mixed with 400 μl of isopropanol. Samples were incubated at − 70 °C overnight to precipitate the RNA. Then the samples were centrifuged at 13 200 rpm (Eppendorf Centrifuge 5415 R) for 15 min at 4 °C, following which RNA pellets were washed with 500 μl of 70% ethanol at 13 200 rpm (Eppendorf Centrifuge 5415 R) for 15 min at 4 °C. RNA pellets were briefly air-dried and resuspended in 20 μl diethyl pyrocarbonate (DEPC)-treated Millipore-purified water. The RNA was purified another round to generate high quality total RNA using the QIAGEN® RNeasy Mini Kit (QIAGEN). The QIAGEN® RNeasy Mini Protocol for RNA Cleanup was followed according to the manufacturers' instructions, with the exception of the final elution of total RNA was performed twice in 10–12 μl volumes of RNase-free water (QIAGEN) with 1 min incubations on the RNeasy® mini column (QIAGEN). RNA concentrations were determined using Nanodrop® ND1000 Spectrophotometer (Nanodrop Technologies). RNA quality was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies) by checking the RNA Integrity Number and examining the electropherogram profile generated.

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