Sybr premix ex taq tli rnaseh plus master mix

Total RNA from each leaf sample was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. cDNA library preparation and transcriptome sequencing follow Liu et al. (2015) and Zhong et al. (2011). Clean reads were mapped to the maize reference genome (B73 RefGen_v3.31) (Schnable et al., 2009) using TopHat2 software (Kim et al., 2013), and only unique mapping reads were retained for calculating gene expression. RNA‐seq data analysis was performed according to previously published protocols (Trapnell et al., 2010, 2012). DEGs were identified by the edge package (http://www. r-project.org/) with FDR <0.05 and absolute value of log2ratio ≥1. To validate the accuracy of the RNA‐seq data, qRT‐PCR analyses were performed on Applied Biosystems 7500 Fast Real‐Time PCR System (Applied Biosysterm, Foster City, CA) using SYBR Premix Ex Taq (Tli RNaseH Plus) master mix (Takara‐Bio, Shiga, Japan) following the manufacturer's instructions. The PCR amplification program was 95 °C (15 s), followed by 40 cycles at 60 °C (60 s) and 95 °C (30 s). Fold changes of gene expression level were calculated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001). The actin gene was used as a candidate reference gene (Manoli et al., 2012). The primers used in this study are given in Table S5.

In order to investigate the effect of sinomenine and celecoxib on P-gp and COX-2 expression, real-time relative quantitative PCR was performed. Cells were plated in 6-well plates with DMEM supplemented with 10% FCS for 24 h. Caco-2 and MDR-Caco-2 cells were treated with sinomenine (500 µM) or celecoxib (25 µM) for 48 h.
Total RNA was isolated with TRIzol reagent (Keygen Biotech Co., Ltd, Nanjing, China), according to the protocol of the manufacturer. The isolated RNA was quantified by spectrophotometry (optical denisty 260/280 nm). The mRNA was then reverse-transcribed into cDNA, according to PrimeScript RT Master Mix Perfect Real Time purchased from Takara Bio Inc. (Dalian, China).
Real-time relative quantitative PCR was performed using the Applied Biosystems 7500 faster Real-Time PCR System with the SYBR Premix Ex Taq (Tli RNaseH Plus) Master Mix purchased from Takara Bio Inc (Dalian, China) in triplicate for each sample and each gene. PCRs were carried out using the oligonucleotide primers listed in Table 1, which describes the size of expected fragments. PCR conditions used were: denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s and 30 s at 60°C for annealing and 30 s at 72°C for elongation. The results were expressed as the ratio value of the CT value for the target mRNA to that of the β-actin mRNA (Ct sample/Ctβ-actin).