Revet aid first strand cdna synthesis kit

The test compounds saikosaponin A (purity > 95%), C (purity > 95%) and D (purity > 95%) were purchased from Winherb Medical Technology (Shanghai, China). Colchicine, rhodamine B, verapamil, and 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RIPA buffer (1×), Pierce^®BCA protein assay kit, and the Trizol^® reagent were from Invitrogen (Carlsbad, CA, USA). The Revet Aid First Strand cDNA Synthesis kit and DyNAmo^TM Color Flash SYBR^® Green qPCR kit were obtained from Thermo Scientific (Rockford, IL, USA). Human Pgp and GAPDH primers were synthesized by Invitrogen (Guangzhou, China). OCT2-pCMV6-AC-GFP, Mrp2-pCMV6-AC-GFP, ABCB1-pCMV6-AC-GFP, and pCMV6-AC-GFP plasmids and Mega Tran1.0 transfection reagent were purchased from OriGene Technologies Company (Maryland, USA). The mouse monoclonal antibodies against organic cation transporter 2 (Oct2), Pgp, Mrp1, and Mrp2 and anti-mouse IgG HRP-conjugated antibody were purchased from Abcam (Cambridge, UK). The GAPDH antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). All other chemicals used in the present study were commercially available and of analytical reagent grade.

Real-Time PCR Analysis of Glutathione metabolism genes
The faba bean leaves stored in liquid nitrogen were utilized for RNA extraction with a Qiagen RNeasy^® Plant mini kit (Qiagen, Hilden, Germany), while first-strand cDNA was synthesized from 0.5 µg of total RNA using a RevetAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA), following the manufacturer’s instructions.
The genes related to the glutathione metabolism pathways (L-APX, SPS, LAP, AP-N, and RDS-M genes) were quantitatively amplified with the aid of StepOnePlus (Applied Biosystems®, Thermo Fisher Scientific, USA) Real-Time PCR system using specific primers (Table 1) and a SYBR^TM Green PCR master mix (Applied Biosystems®, Thermo Fisher Scientific, USA). The transcription level of the Vicia faba Elongation Factor-alpha (VfEFα) gene was used as an endogenous control, to which the transcription level of the addressed genes were normalized using the 2^−DDCt method. The amplification protocol consisted of 40 amplification cycles (95 °C for 15 s and 60 °C for 60 s) preceded by 95 °C for 10 min. For each gene, expression estimated in Ctrl was used as a quantification unit.

Total RNA from HUVECs was extracted using the RiboEX reagent (Invitrogen). One microgram of total RNA was reverse-transcribed using the Revet Aid First Strand cDNA Synthesis kit (Thermo, Rockford, IL, USA), according to the manufacturer’s instructions. Real-time PCR was performed using the SYBR Premix Ex Taq (Takara, Shiga, Japan), following the manufacturer’s protocol. All reactions were performed in triplicates. The cDNA was amplified using 60 cycles of 15 s at 95 °C, 15 s at 60 °C, and 30 s at 72 °C for each gene. The expression values are presented relative to the β-actin values in the corresponding samples. The primers used in this study are shown in Supplementary Table S1.