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 RNA was isolated using TRIzol (Gibco, Invitrogen, Grand Island, NY, USA) and purified using RNeasy Mini kit (Qiagen, Hamburg, Germany). The RNA concentration was quantified at 260 nm and 1 μg of RNA was transcribed using SuperScript II (Invitrogen, Carlsbad, CA, USA) reverse transcriptase and oligo(dT) primers (Invitrogen). Quantitative RT-PCRs were performed in duplicates by iCycler iQ™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) in 30 μl reaction volume containing 1X iQ™ SYBR Green Supermix (0.2 mM dNTP, 3 mM MgCl2), 0.5 μM forward and reverse primer and 2 μl of cDNA. The reaction conditions were: 95°C 3 min, 40 cycles (94°C 30 sec, 55°C 50 sec, 72°C 50 sec), 72°C 7 min followed by melting curve analysis to confirm amplification of the desired single and specific product. The relative expression levels of hypoxia-inducible factor-1α (HIF-1α), Epo, VEGF and β-actin genes (primers designed by SABiosciences) were evaluated using the standard curve method. Standard curves for HIF-1α, Epo, VEGF and β-actin were obtained by amplification of serially-diluted mixtures of cDNA samples (four-fold dilutions), with four to five dilution points, each in duplicate. The calculated resulting relative expression of HIF-1α, Epo, VEGF genes were normalized to relative β-actin expression. The results are presented as mean ± standard deviation of three independent experiments.

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Manufacturer protocol

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