Hybrid r rna extraction kit

Hesperetin was obtained from Sigma-Aldrich (W431300, Germany). Cell culture reagents, including culture medium (L0093) and antibiotics (L0022) were obtained from Biowest (France). Fetal bovine serum (FBS) was purchased from Biosera (FB-1001/100, France). Hybrid-R RNA extraction kit (305 − 101, GeneAll Biotechnology, Korea) was used for the isolation of total cellular RNA and the corresponding cDNA was synthesized with HyperScript™ RT Master Mix (601–710, GeneAll Biotechnology, Korea). RealQ Plus 2x SYBR Green Master Mix high ROX™ (A325402, Ampliqon, Denmark) was used for real-time PCR. BCA Protein Assay Kit (23,225) was purchased from Thermo Fisher Scientific, UK). RIPA cell lysis buffer (PL008-5X) was attained from Biobasic (Canada). All the chemicals, including MTT (M5655), dimethyl sulfoxide (DMSO) (67-68-5), and general laboratory reagents were obtained from Sigma-Aldrich (Germany).

Total RNA was extracted using a Hybrid-R RNA extraction kit (GeneAll Biotechnology, Seoul, South Korea). cDNA was synthesized by M-MLV cDNA Synthesis kit (Enzynomics, Daejeon, South Korea) according to the supplier’s instructions. Quantitative real-time PCR was performed using TOPrealTM qPCR 2X PreMIX (Enzynomics) on a CFX Connect Real-Time PCR Detection system (Bio-Rad Laboratories, Hercules, CA, USA). Primers used were 5’-AGGGCATCATCAATTTCGAG-3’ (sense) and 5’-TGCCTCTCTTCATCCTTTGG-3’ (antisense) for human SOD1 (NCBI gene ID: 6647); 5’-GTGTGATGGTCCTTCCAACC-3’ (sense) and 5’-CTGACATCCTCTGGCTCACA-3’ (antisense) for human Prdx6 (NCBI gene ID: 9588); 5’-CAGTCTCAAGTATGTCCGT-3’ (sense) and 5’-AGGCTCAATGTTGATGGT-3’ (antisense) for human Gpx2 (NCBI gene ID: 2877); 5’-TTGGCTACCTTGCAGTTCGT-3’ (sense) and 5’-ATGTGAACCATCGCAGGCA-3’ (antisense) for human CISD2 (NCBI gene ID: 493856); 5’-CATGTACGTTGCTATCCAGGC-3’ (sense) and 5’-CTCCTTAATGTCACGCACGAT-3’ (antisense) for human β-actin (NCBI gene ID: 60). Ratio of target gene fold-change was normalized to human β-actin expression using comparative CT (2-ΔΔCt) method.

Total RNA was isolated using a Hybrid-R RNA extraction kit (GeneAll, Seoul, Korea). Total RNA (1 μg) was subject to cDNA synthesis, using PrimeScript RT-PCR kit (TaKaRa Korea, Seoul, Korea). Real-time PCR was performed on a CFX96 instrument (Bio-Rad, Hercules, CA, USA) using EvaGreen Supermix (Bio-Rad, Hercules, CA, USA). The primer sequences used for the quantitation of target genes are illustrated in Table 1. GAPDH was used as an internal control.

Total RNA was extracted using a Hybrid-R RNA extraction kit (GeneAll, Seoul, Republic of Korea). Synthesis of cDNA from total RNA (1 μg) was conducted using AmfiRivert cDNA Synthesis Platinum master mix (GenDEPOT, Austin, TX, USA). Real-time RT-PCR analysis was performed using SYBR mix (ELPIS Biotech, Daejeon, Republic of Korea) on the CFX384 real-time system as recommended by the manufacturer (BioRad, Hercules, CA, USA), and the mRNA level of individual genes was normalized by that of GAPDH. PCR primer sequences against individual genes are listed (Table 1).

Total RNA was extracted from the MSCs using Hybrid-R™ RNA Extraction Kit (GeneAll, Seoul, South Korea), followed by treatment with DNase I (RNase-free; Invitrogen, Carlsbad, CA, USA) at 37 °C for 15 min to eliminate DNA contamination. Reverse transcription reactions were performed to synthesize cDNA using RevertAid M-MuLV Reverse Transcriptase Kit (Fermentase, Hanover, Germany) according to the manufacturer’s instructions. RT-qPCR was carried out with the SYBR Green I Master Mix kit (Ampliqon, Copenhagen, Denmark) using the ABI 7500 System (Applied Biosciences, Foster City, CA). The copies of target genes were normalized to reference gene GAPDH RNA level as the internal normalization control. Relative expression was calculated using the 2-ΔΔct method. Sequences of Primers are provided in Supplementary Table 1.