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

Isolated FRT tissues were chopped into small pieces, collected in TRIzol tubes filled with 3 mm of zirconium beads, and homogenized in three 10-s pulses using a Beadbug microtube homogenizer (Benchmark Scientific). RNA was isolated with TRIzol reagent according to the manufacturer’s instructions (Invitrogen). 1–5 µg RNA was reverse transcribed into cDNA with a Maxima First Strand cDNA Synthesis kit with dsDNase (Thermo Fisher Scientific). Real-time PCR was performed using 2x SensiFAST probe Hi-Rox mix (Bioline) with gene-specific primers (Table S1) and run on an ABIPrism 7900 sequence detector with the ΔΔCt method from SDS 2.4 software (Applied Biosystems). The relative expression of genes was calculated with the formula 2−ΔCt, where ΔCt = Ct target gene − Ct endogenous control gene. Gapdh was used as the endogenous control housekeeping gene. Viral burden was determined from a standard curve produced using serial 10-fold dilutions of ZIKV RNA or LCMV plasmid.

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Manufacturer protocol

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