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

Total cellular RNA was isolated using TRI Reagent (Sigma-Aldrich) according to manufacturer's protocol. Complementary DNA was synthesized using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific) with 4 μg of RNA used per reaction. PCR amplification was performed in 384-well microplates at 10 μl of total volume, containing cDNA, 0.5 μM primer mix (primer sequences in Supplementary Table 1) and LightCycler 480 SYBR Green I Master Mix (Roche Applied Science, Penzberg, Germany). Real time PCR amplifications were performed in a LightCycler 480 (Roche) under the following conditions: 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 20 s. Three reference (constitutive expression) genes were selected using the GeNorm procedure [51]: HPRT1, HMBS and TBP. Expression of assayed genes was expressed as number of cognate mRNA copies per 1000 copies of geometric-averaged reference mRNA.

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for TRI Reagent® Sigma below.

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