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

Total or fractionated RNA was extracted using TRIzol (Invitrogen/Life Technologies, Carlsbad, CA) and treated with DNaseI (Roche, Mannheim, Germany). RNA was reverse transcribed using RevertAid H Minus Reverse Transcriptase (Fermentas, St Leon-Rot, Germany). To test mature miRNA levels, gene-specific RT was performed using stem-loop primers shown in Table W1 according to Chen et al. [44]. For the identification of Drosha splice variants, complementary DNA (cDNA) of different cell lines was used as a template to amplify exons 29 to 35 of Drosha. Polymerase chain reaction (PCR) products were visualized on a 1.2% agarose gel using ethidium bromide. Bands that ran lower than the expected wild-type band were purified from Ma-Mel-71, Ma-Mel-75, and NT2; cloned into pCRII-TOPO (Invitrogen) by TOPO-TA cloning; and sequenced.

Quantitative PCR was performed on an ABI StepOne Plus using 2x Power SybrGreen MasterMix (Applied Biosystems/Life Technologies).

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Manufacturer protocol

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