Taqman gene expression assay sets

Total RNA was isolated using Trizol (15596018, Carlsbad, CA, USA, Thermo Fisher Scientific). The concentration of RNA was determined spectrophotometrically (BioTek). Four micrograms of RNA was reverse-transcribed into cDNA using ReverTra Ace^® reverse transcription kit (Toyobo, FSQ101, Osaka, Japan). The reverse transcription was stopped by adding Tris–EDTA buffer (pH 8.0) to a total of 200 μL of cDNA solution. The TaqMan^® Gene Expression Assay sets were purchased from Applied Biosystems. Q-RT-PCRs were done according to the manufacturer’s instructions. Briefly, 20 μL of Q-PCR mixture contained 10 μL 2× TaqMan Universal Master Mix (Applied Biosystems, Foster City, CA, USA), 1 μL 20× Taqman expression assay (Applied Biosystems), and 50 ng cDNA. Q-PCR mixture was reacted in 95 °C for 20 s and 40 times cycling reaction (95 °C for 1 s and 60 °C for 20 s) with a StepOnePlus System (Applied Biosystems). The gene identification numbers for the TaqMan expression assay used in the Q-RT-PCR analyses are presented in Table 1. Human GAPDH (Applied Biosystems) was used for normalizing variation in cDNA quantities from different samples.