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 from cell lines was extracted by
using the TRIzol reagent (Invitrogen) and DNase treated with RQ1 DNase (Promega). For mRNA expression analysis, purified total RNA was reverse-transcribed by using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). Real-time
PCRs were performed in triplicate in an Applied Biosystems 7900HT Fast Real-Time PCR system, using 100 ng of cDNA, 6 μL of SYBR Green PCR Master Mix (Applied Biosystems) and 416 nM primers (listed in Table S1) in a final volume of 12 μL for
384-well plates. All data were normalized with respect to two housekeeping genes (L13 and PBGD), with no significant GC skew at their promoters as endogenous control. Relative RNA levels were calculated by using the comparative Ct method (ΔΔCt), considering the PCR efficiency. The order of magnitude of change
is equal to 10ΔΔCt/m, where m is the average slope of the calibration curves for the gene of interest and the endogenous control.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for TRIzol Reagent below.

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