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

Growing cells were harvested, cell pellets washed with phosphate buffered saline (PBS) and total RNA extracted using Trizol (Invitrogen, Paisley, UK) according to standard procedures. For each cell line 2 µg of RNA was reverse transcribed into cDNA using Omniscript reverse transcriptase (Qiagen). Briefly, the RNA was incubated for 60 min at 37 °C with reverse transcription reaction mixture containing 0.5 mM of each dNTP, 1 µM oligo-dT primer (Eurogentec SA, Seraing, Belgium), 10 units RNase inhibitor (Applied Biosystems, Foster City, CA, USA) and 4 units Omniscript reverse transcriptase (Roche, Applied science, Indianapolis, USA) in 20 µL total reaction volume. Human tumor biopsies: we used 10 sections (10 µm thickness) of the frozen biopsies for RNA extraction and subsequent cDNA synthesis. Frozen sections were homogenized using a Qiashredder homogenizer and total RNA was subsequently extracted using the RNeasy Micro kit (Qiagen). cDNA synthesis was performed using the First strand cDNA synthesis kit (Fermentas, St Leon-Rot, Germany). Quality control of the newly synthesized cDNA was done using a human β-actin-based PCR amplification reaction.

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Manufacturer protocol

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