Syber green 1 step qrt pcr kit

Rutin (RU), gallic acid (GA), catechin, ursolic acid (UA), Folin–Ciocalteau reagent, 2,2 diphenyl-1-picrylhydrazyl (DPPH), 2,4 dinitrophenyl hydrazine (DNPH), 5, 5′, dithiobis-2-nitrobenzoic acid (DTNB), butylated hydroxytoluene (BHT), CCl₄ (reagent grade, 99.9%), 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), NADPH, and GSH were acquired from Sigma-Aldrich, St Louis, MO, USA. Thiobarbituric acid (TBA) was gained from El-Nasr Pharmaceutical Chemicals Co. (Alex., Egypt). Ascorbic acid [(Asc): vitamin C] and Trolox were bought from Riedel-de Haën, Germany. Biozol reagent was purchased from Invitrogen, CA, USA. SYBER Green 1-step qRT-PCR Kit was purchased from Thermo Scientific, USA. Primers for tumor necrosis factor (TNF)-α, nuclear factor-kappa B (NF-κB), transforming growth factor (TGF)-β1, interleukin (IL)-6, and the tumor suppressor gene p53 were acquired from Bioneer, Korea. Kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin, creatinine, urea, triglycerides (TG), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) were gained from Biodiagnostic, Cairo, Egypt.

Folin-Ciocalteau reagent, gallic acid (GA), ursolic acid (UA), rutin (RU), catechin, 2,4 dinitrophenyl hydrazine (DNPH), 2,2 diphenyl-1-picrylhydrazyl (DPPH), butylatedhydroxytoluene (BHT), 5, 5′, Dithiobis-2-nitrobenzoic acid (DTNB), 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), CCl₄ (reagent grade, 99.9%) and GSH were obtained from Sigma-Aldrich, St Louis, MO, USA. thiobarbituric acid (TBA) was obtained from El-Nasr Pharmaceutical Chemicals Co. (Alex., Egypt). Ascorbic acid (Asc) was obtained from Riedel-de Haën, Germany. Biozol reagent was obtained from Invitrogen, CA, USA. SYBER Green 1-step qRT-PCR Kit was purchased from Thermo Scientific, USA. Primers for nuclear factor kappa B (NF-κB), tumor necrosis factor (TNF)-α, interleukin (IL)-6, transforming growth factor (TGF)-β and the tumor suppressor gene p53 were purchased from Bioneer, Korea. Kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin, creatinine, urea, high-density lipoprotein cholesterol (HDL-c) low-density lipoprotein cholesterol (LDL-c) and triglycerides (TG) were purchased from Biodiagnostic, Cairo, Egypt.

Total RNA from bladder mice’s tissue, was isolated using Gene JET RNA Purification Kit (Thermo Scientific, USA). The extracted RNAs from different samples were quantified and qualified (purity) using NanoDrop Spectrophotometer. Total RNAs samples were normalized (same concentration) to avoid any false increase in gene expression levels. Using SYBER Green 1-step qRT-PCR Kit (Thermo Scientific, USA), gene expression of TNF-ɑ (target gene) and β-actin (reference gene) were quantified by Real-Time PCR System (Thermo Scientific PikoReal) with the use of specific primers sequences (Forward/Reverse)forward 5′-CATCTTCTCAAAATTCGAGTGACAA-3′, reverse 5′-TGGGAGTAGACAAGGTACAACCC-3′ for TNF-ɑ oncogene [17 (link)]. and 5′-GCTGTATTCCCCTCCATCGT-3′/5′-GAGTCCTTCTGACCCATTCC-3’ for β-actin gene [18 (link)]. qRT-PCR was performed in a reaction mixture of 10 μl using 0.1 μl verso enzyme mix, 5 μl 1-step QPCR SYBER mix (1X), 0.5 μl RT-enhancer, 0.5 μl forward and reverse primers (10 pm), 0–2.9 μl water (PCR grade) and 0.5–3.4 μl RNA template (50 ng). qRT-PCR program was applied as one cycle of cDNA synthesis at 50 °C for 15 min, one cycle of Thermo-start enzyme activation at 95 °C for 15 min and followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min and extension at 72 °C for 30 s.