TRIzol Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Protocol tips
- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Publication protocol

For miRNA profiling, about 2 × 106 cells were harvested in 1.5 ml Trizol (Life Technologies), homogenized, mixed with 0.45 ml chloroform and phase separated by centrifugation. RNA was precipitated by aspiration of the upper aqueous phase, addition of 1.25 ml isopropanol, mixing and centrifugation. The pellet was washed with 75% ethanol, then dried and dissolved in water. Total RNA concentrations were determined photometrically using the NanoDrop 1000 ND-1000 (Peqlab, Erlangen, Germany). Overall RNA quality was assessed on a 1% agarose gel with a 1 KB molecular weight marker separated in parallel. Cells from MEK-inhibitor experiments were lyzed with 700 μl Qiazol (Qiagen, Hilden, Germany), thoroughly mixed with 140 μl chloroform and centrifuged at 12,000 × g at 4°C for 15 minutes. The upper aqueous phase was mixed with 1.5 volumes of ethanol and loaded onto an RNeasy Mini spin column (RNeasy Mini Kit, Qiagen). RNA extraction was performed according to the manufacturer’s instructions.

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Manufacturer protocol

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