Agilent mx3005p real time pcr system

The total RNA was extracted from tissues or cultured cells with Trizol reagent (Invitrogen) and reverse transcribed with PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa). Quantitative PCR was performed with SYBR green (SYBR Premix Ex Taq, TaKaRa) `at Agilent Mx3005P real-time PCR system (Agilent Technologies). Expression analysis in different cell types was performed using specific primers for each gene.

All RNA from liver tissues was isolated using the Promega SV Total Isolation System (Promega, Madison, WI, United States) and quantified using a microplate reader. cDNA was synthesized using the PrimerScript first strand cDNA Synthesis Kit (Takara, Shiga, Japan). The mRNA expressions of key enzymes (CYP27A1, CYP7A1, and CYP8b1), transport (NTCP, bile salt excretion pump (BSEP), MRP2, MRP3, and MRP4), and metabolic enzymes (CYP) were detected using the Agilent Mx3005 P Real-Time PCR System (Agilent, Santa Clara, CA, United States) with the SYBR Green PCR kit. The thermal conditions were as follows: 95°C for 60 s, followed by 45 cycles at 95°C for 15 s and 60°C for 60 s. β-Actin was used as an internal control. All primers used for qRT-PCR were synthesized by Sangon Biotech (Shanghai, China) and are presented in appendix, section 3 (Supplementary Appendix S3).

Total RNA of clinical blood and U251 cell line samples were extracted using the TRIzol™ reagent according to TRIZOL method (Cat. No: 15596026, Invitrogen, USA). Quality and integrity of RNA were determined through the NanoDrop®ND-1000 UV–Vis Spectrophotometer (NanoDrop Technologies, Wilmington, USA) and agarose gel electrophoresis. The first-strand cDNA was synthesized from 1 μg of total RNA using SuperScript® III Reverse Transcriptase Kit (Cat. No: 18080044, Invitrogen, USA) following the protocol. All of qPCR reactions were performed through SYBR Green-based methods together with the GoTaq® qPCR Master Mix (Cat. No: A6001, Promega, USA) and primers on Agilent Mx3005P Real-Time PCR system (Agilent, USA). 1 μl cDNA was used in a total volume of 20 μl, with a primer concentration of 200 nM. Every reaction was initiated at 10 min at 95°C, followed by 40 cycles consisting of 15 s denaturation at 95°C, 60 s annealing at 60°C and 30 s extension at 72°C. A final dissociation stage was run to generate a melting curve for verification of amplificated product specificity. The relative mRNA expression levels with respect to the housekeeping gene (GAPDH) were measured using the 2^−ΔΔCT method. All qPCR experiments were performed in triplicate. Primer pairs were designed with the Primer Premier 5 software (Premier Biosoft International, Palo Alto, CA) and validated by iPAGE.