Real time pcr kit

PQ was purchased from Sigma Chemical Co. (Sigma-Aldrich, Milan, Italy). ReNcell NSC Maintenance Medium and accutase were obtained commercially from Millipore (Temecula, CA). Epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-2) were purchased from PeproTech. Laminin was purchased from Invitrogen (Carlsbad, CA, USA). Catalase Assay Kit, Malondialdehyde Assay Kit, Lactate Dehydrogenase Assay Kit, BCA Protein Assay Kit, Cell Lysis Buffer for Western and IP, goat anti-rabbit IgG-HRP, and goat anti-mouse IgG-HRP were obtained from Beyotime (Jiangsu, China). Total Superoxide Dismutase Assay Kit was purchased from Dojindo Molecular Technologies (Kumamoto, Japan). Tripure was obtained from Roche (Basel, Switzerland). The AMV first strand cDNA Synthesis Kit was purchased from MBI (Fermentas, Canada). Real-time PCR Kit was obtained from Tiangen Biotech (Beijing, China). Rabbit anti-Nrf2 polyclonal antibody, rabbit anti-Keap1 polyclonal antibody, rabbit anti-PKC polyclonal antibody, and rabbit anti-CKII polyclonal antibody were purchased from GeneTex (San Antonio, USA). Mouse anti-β-tubulin polyclonal antibody was purchased from Boster (Wuhan, China).

Cultured cells were washed once with ice-cold PBS and collected with TRIzol reagent (Thermo Fisher Scientific, USA). Total RNA of cells was isolated with chloroform. The quantity and concentration of RNA were evaluated with a NanoDrop system (Thermo Fisher Scientific, USA). A total of 1 μg RNA was reverse transcribed into cDNA with PrimeScript™ RT Master Mix (TaKaRa, Japan) [16 (link)]. The mRNA levels of the genes were quantified with a real-time PCR kit (Tiangen, China) on a Bio-Rad CFX96 real-time PCR system according to the manufacturer’s instructions. GAPDH was used as the internal control. The sequences of specific primers used in this study are listed below: LIMD2-F: 5′-CAGGAAGACCCTACCAAATATC-3ʹ LIMD2-R: 5ʹ- CCCAACAGGGCTGATTTAC-3ʹ GAPDH-F: 5ʹ-GTATGACAACAGCCTCAAGAT-3ʹ GAPDH-R: 5ʹ-GTCCTTCCACGATACCAAAG-3ʹ. Three biological replicates were performed.

Total RNA was purified from tissue and cell specimens with TRIzol reagent (Invitrogen) as directed by the manufacturer. Reverse transcription was carried out with reverse transcription kit (K1622; Thermo Scientific, USA), and the Real-Time PCR kit (FP209-02; TIANGEN, China) was used for qRT-PCR on an ABI 7500-Fast Real-Time PCR System (Applied Biosystems, USA). The data were normalized to β-actin expression, and analyzed by the 2^−ΔΔCt method. Primers are described in Table 1.