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 TrizolTM (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The concentration of RNA was determined spectrophotometrically, and the integrity of the RNA was assessed using a BioAnalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Two micrograms of RNA were reverse-transcribed into cDNA using SuperScript®III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and aliquots were stored at −20℃. Quantitative real-time TaqMan RT–PCR technology (Q-RT-PCR) (Applied Biosystems, Foster City, CA, USA) was used to determine the expression level of selected target genes. The cycling conditions included a denaturing step at 95℃ for 10 min and 50 cycles of 95℃ for 15 sec and 60℃ for 1 min. The TaqMan probes (Applied Biosystems) used in the qRT-PCR analysis were: Filaggrin, Hs00856927_g1; Loricrin, Hs01894962_s1; Involucrin, Hs00846307_s1; TGM1, Hs01070316_m1; TGM2, Hs01096681_m1; KRT10, Hs01043110_g1; and HBD2, Hs00175474_m1. Human GAPDH (4333764F, Applied Bio-systems) was also amplified to normalize variations in cDNA quantities from different samples. Relative differences in gene expression were calculated from Ct (threshold cycle) values using equations from a mathematical model developed by Pfaffl

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Manufacturer protocol

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