RNA extraction and quantitative real-time reverse transcription polymerase chain reaction (q-RT-PCR)

Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The RNA concentration was spectrophotometrically determined, and RNA integrity was analyzed using a BioAnalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA); 4 μg were reverse transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen). The expression levels of target genes were evaluated by qRT-PCR using TaqMan technology (7500 Fast; Applied Biosystems, Foster City, CA, USA). The cycling protocol was as follows: 95 °C for 10 min, followed by 50 cycles of 95 °C for 15 s and 60 °C for 1 min. The target genes were as follows: keratin 1, Hs00196158_m1; keratin 10, Hs00166289_m1; IFT88, Hs00544051_m1; and SPRR3, Hs00271304_m1. Ribosomal protein L13A (Hs04194366_g1; Applied Biosystems) was used to normalize the expression levels of target genes.