Rox reference dye

Glyoxalase I expression vs β-actin was evaluated by quantitative real-time TaqMan PCR analysis (qRT–PCR) on a MX3000P Real-Time PCR System (Agilent Technology). The sequences of oligonucleotide primers and TaqMan probes used for qRT–PCR were as follows: GI: 5′-CTCTCCAGAAAAGCTACACTTTGAG-3′ (sense, 400 nM), 5′-CGAGGGTCTGAATTGCCATTG-3′ (antisense, 400 nM), 5′-FAM-TGGGTCGCATCATCTTCAGTGCCC-TAMRA-3′ (TaqMan Probe, 200 nM); β-actin: 5′-CACTCTTCCAGCCTTCCTTCC-3′ (sense, 600 nM), 5′-ACAGCACTGTGTTGGCGTAC-3′ (antisense, 600 nM), 5′-TEXASRED-TGCGGATGTCCACGTCACACTTCA-BHQ-3′ (TaqMan Probe, 200 nM). All PCR primers and probes were designed using Beacon Designer 4 software (Stratagene, Santa Clara, CA, USA), starting from published sequence data supplied by the NCBI database. The PCR reactions were performed in a total volume of 25 μl, containing 250 ng of cDNA, 1 × Brilliant QPCR master mix (Agilent Technology), 0.5 μl of ROX Reference Dye (Agilent Technology) and a concentration of specific primers and probes. The thermal cycling conditions were as follows: 1 cycle at 95 °C for 10 min, followed by 45 cycles at 95 °C for 20 s and 55 °C for 1 min. Data for comparative analysis of gene expression were obtained using the ΔΔCt method (Livak and Schmittgen, 2001 (link)).