Myiq rt pcr system

The expression levels of cytochrome P450 genes were quantified by quantitative real-time PCR (qRT-PCR) using an MYiQ’ RT-PCR system Bio-Rad, California, USA) and Real Master Mix 2xSYBR Green qPCR mix (Aidlab Biotechnologies Co., Ltd, China). QRT-PCR of each cDNA sample and template-free was performed in triplicate. Specific primers of CYP6AE10, CYP9A11, and CYP321A8 were designed for qPCR (Table 2). Reaction volume of 20 µL was used (0.5 µL of each primer 10 µM, 1 µL cDNA, 8 ul ddH20 and 10 µL 2x cyber master mix for quantification using the following cycling parameters: 94 °C for 3 min, followed by 40 cycles of 94 °C for 15 sec, 57–60 °C for 30 s and 70 °C for 30 s. For each gene, a serial dilution from 10- to 1000-fold of each cDNA template was performed in order to assess the efficiency of PCR. The relative expression values were calculated using the 2^−ΔΔCT methods as previously described by Livak and Schmittgen (2001) . Results were expressed as the mean expression ratio (±S.E.) of three biological replicates between chemical treatments and controls. One-way analysis of variance (ANOVA) and the Tukey HSD test for the significant difference was performed to determine the statistical difference between means (SPSS, version 19).

Main experimental instruments included a cell culture incubator maintaining constant temperature (SANYO Inc., Okayama, Japan), a Centrifuge 58108 (Eppendorf Inc., Hamburg, Germany), an LX-200 mini centrifuge (Haimen Kylin-Bell Lab Instruments Co., Ltd., Jiangsu, People’s Republic of China), a TC automatic cell counter (Bio-Rad Inc., Hercules, CA, USA), an electrophoresis apparatus (Beijing Liuyi Biotechnology Co., Ltd, Beijing, People’s Republic of China) and electrophoresis tank (Bio-Rad Inc.), a gel-imaging analyzer (Alpha Inc., USA), a semiquantitative gradient polymerase chain reaction (PCR) system, MyiQ RT-PCR system (Bio-Rad Inc.), a NanoDrop 2000 trace oxygen meter (BioTek Inc., Winooski, VT, USA), a synergy 2 multifunctional microplate reader (BioTek Inc.), and an inverted fluorescence microscope (Olympus Inc., Tokyo, Japan).