TRI Reagent® Sigma

Protocol tips

Upstream tips	Protocol tips	Downstream 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.

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

High pure total-RNA isolation kit (Roche, Basel, Switzerland) was used for RNA isolation and high pure miRNA isolation kit for miRNA isolation. For both isolations the medium was removed and samples were twice washed with 5 ml of ice-cold PBS. Cells were scraped off, transferred to clean tubes and centrifuged at 20,800 × g for 5 min at 4°C. After this step, lysis buffer was added and RNA isolation was carried out according to manufacturer’s instructions. Isolated RNA and miRNA was used for cDNA synthesis. RNA (600 ng) was transcribed using Transcriptor first strand cDNA synthesis kit (Roche, Switzerland) and 10 ng of miRNA was transcribed using TaqMan® microRNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA) according to manufacturer’s instructions. Prepared cDNA (20 μl) from total-RNA was diluted with RNase-free water to 100 μl and 5 μl was directly analyzed by 7500 real-time PCR system (Applied Biosystems). In case of miRNA analysis 1.33 μl of transcribed miRNA was used directly in real-time PCR reaction according to manufacturer’s instruction

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Manufacturer protocol

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