Topreal qpcr 2 premix sybr green

Materials were purchased respectively as follows: EGM-2 medium kit from Lonza Cambrex (Nottingham, UK), enhanced chemiluminescence (ECL) reagent from AbClon (Seoul, South Korea), Griess reagent from Promega Co. (WI, USA), LeGene Premium Express 1st Strand cDNA Synthesis System from LeGene Biosciences (CA, USA), polyvinylidene fluoride (PVDF) membranes from Millipore (MA, USA), pyridine-d5 from Cambridge Isotope Laboratories Inc. (MA, USA), RNAiso PLUS from TAKARA Korea Biomedical Co. (Seoul, South Korea), thin-layer chromatography (TLC) silica gel 60 F₂₅₄ from Merck (Darmstadt, Germany), and TOPreal™ qPCR 2× PreMIX SYBR green from Enzynomics (Seoul, South Korea). N(G)-nitro-L-arginine methyl ester (L-NAME), fetal bovine serum (FBS), and silica gel resin were purchased from Sigma-Aldrich (MO, USA). All other chemicals were of ultra-pure grade. The primary antibodies (eNOS, phospho-eNOS Ser¹¹⁷⁷, Akt, phospho-Akt Thr³⁰⁸, and GAPDH) and horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit and anti-mouse) were obtained from Merckmillipore (CA, USA). All other chemicals were of ultra-pure grade.

mRNA transcription of cytokines was analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Using the Hybrid-R™ (GeneAll, Seoul, Korea), we extracted total RNA from the BV2 microglial cells, and measured the concentration using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Next, 3 μg RNA samples were converted to cDNA using TOPscript™ RT DryMIX. The cDNA was analyzed by qRT-PCR using TOPreal™ qPCR 2× PreMIX (SYBR Green; Enzynomics) and the CFX Connect Real-Time PCR System (Bio-Rad Laboratories, CA, USA). Primers, synthesized at COSMO Genetech (Seoul, Korea), were as follows; iNOS: forward, 5’-GTGTTC TTTGCTTCCATG CT-3’, reverse, 5’-AGTTGCTCCTCTTCCAAG GT-3’; TNF-α: forward, 5’- GAGTGACAAGCCTGTAGCCCA-3’, reverse, 5’- AGCTCCACGCCATTGGC-3’; IL-1β: forward, 5’-CCCAAGCAATACCCA AAG AA-3’, reverse, 5’-GCT TGTGCTCTGCTTGTGAG-3’; IL-10: forward, 5’-CTAGAGCTGCGGACTGCCTTC-3’, reverse, 5’-TTGATTTCTGGGCCATGC-3’; COX2: forward, 5’-TCATTG GTGGAGAGGTGTAT-3’, reverse, 5’-ACCCCACTCAGGATGCTCCT-3’; GAPDH: forward, 5’-TGA ATACGGCTACAGCAACA-3’, reverse, 5’- AGGCCCCTCCTGTTATTATG-3’.IL-6: forward, 5’-TAGTCCTTCCTACCCCAATTTCC-3’, reverse, 5’- TTGGTCCTTAGCCACTCCTTC-3’; IFN-γ: forward, 5’- TGTTACTGCCACGGCACAGT-3’, reverse, 5’- CTGGCTCTGCAGGATTtpTTCAT -3’.

NCI-H441 (2.5 × 10⁵ cells/mL) cells were cultured in six-well plates and pretreated with CA (5 and 10 μM) or rotenone (5 μM) for 24 h, before being treated with UPM (100 μg/mL) in conditioned medium for 6 h. Total RNA was extracted from cells using RNAiso PLUS (TAKARA Korea Biomedical Co, Seoul, Korea). cDNA was generated using LeGene Premium Express First Strand cDNA Synthesis System (Legene, Sandiego, CA). Real-time PCR was performed with TOPreal™ qPCR 2× PreMIX SYBR green (Enzynomics, Seoul, Korea) and analyzed using an iQ5 Thermal Cycler (Bio-Rad, CA, USA). Primers are listed in Table 1. mRNA expression levels were normalized to that of β-actin. Products were analyzed in 1.0–1.5% agarose gels under UV light.Table 1

Real-time PCR primer sequences

Gene	Oligonucleotide sequence	Size
TNF-α	sense, 5’-CCCAGGGACCTCTCTCTAATCA-3’antisense, 5’-GCTACAGGCTTGTCACTCGG-3’	80 bp
IL-6	sense, 5’-GGTACATCCTCGACGGCATCT-3’antisense, 5’-GTGCCTCTTTGCTGCTTTCAC-3’	81 bp
IL-8	sense, 5’-AGAGTGATTGAGAGTGGACC-3’antisense, 5’-ACTTCTCCACAACCCTCT-3’	118 bp
ZO-1	sense, 5’-GTGTTGTGGATACCTTGT-3’antisense, 5’-GTGTTGTGGATACCTTGT-3’	92 bp
Occludin	sense, 5’-GAAGCCAAACCTCTGTGAGC-3’antisense, 5’-GAAGACATCGTCTGGGGTGT-3’	210 bp
β-Actin	sense, 5’-AGCGAGCATCCCCCAAAGTT-3’antisense, 5’-GGGCACGAAGGCTCATCATT-3’	285 bp

ZO-1 zonula occludens, TNF- α tumor necrosis factor-α, IL-6 and-8 interleukin-6 and 8