Sybr green fluorescent label

Real-time qPCR was performed using a CFX96 C1000 (Bio-Rad, Hercules, CA, USA) detection system with SYBR green fluorescent label (Bio-Rad). Samples (25 μl final vol) contained the following: 1× SYBR green master mix (Bio-Rad), 5 pmol of each primer and 0.25 ml of the reverse transcriptase (RT) reaction mixture. Samples were run in triplicate in optically clear 96-well plates. Cycling parameters were as follows: 50 °C × 2 min, 95 °C × 10 min, then 40 cycles of 95 °C × 15 s, 60 °C × 1 min. A melting temperature-determining dissociation step was performed at 95 °C × 15 s, 60 °C × 15 s and 95 °C × 15 s at the end of the amplification phase. The 2^−ΔΔCt method was used to determine the relative gene expression (Livak and Schmittgen, 2001 (link)). The GAPDH gene was the internal control for all qRT-PCR experiments. Data from each group (n = 3) were averaged and shown as normalized gene expression ± SD. One-way ANOVA and Holm–Sidak pair-wise multiple comparison post-hocs (Sigma Stat) were performed. Statistical significance (*) was based on P < 0.05.

Real-time qPCR was performed using a CFX96 C1000 (Bio-Rad, Hercules, CA, USA) detection system with SYBR green fluorescent label (Bio-Rad). Samples (25 μl final volume) contained the following: 1 × SYBR green master mix (Bio-Rad), 5 pmol of each primer and 0.25 μl of the reverse transcription reaction mixture. Samples were run in triplicate in optically clear 96-well plates. Cycling parameters were as follows: 50°C 2 min, 95°C 10 min, then 40 cycles of 95°C 15 s, 60°C 1 min. A melting temperature-determining dissociation step was performed at 95°C 15 s, 60°C 15 s and 95°C 15 s at the end of the amplification phase. The 2ΔΔCt method was used to determine the relative gene expression (Livak & Schmittgen, 2001 (link)). The GAPDH gene was the internal control for all qPCR experiments. Data from each group (n = 3) were averaged and shown as normalized gene expression with SD. One-way ANOVA (Sigma-Stat) and Holm-Sidak pairwise multiple comparison post-hoc analyses were used to determine statistical significance. Statistical significance was based on P < 0.05.

Real-time qRT-PCR was performed using a CFX96 C1000 (Bio-Rad, Hercules, CA, USA) detection system with SYBR green fluorescent label (Bio-Rad). Samples (25 µl final vol) contained the following: 1 × SYBR green master mix (Bio-Rad), 5 pmol of each primer, and 0.25 µl of the reverse transcriptase (RT) reaction mixture. Samples were run in triplicate in optically clear 96-well plates. Cycling parameters were as follows: 50°C × 2 min, 95°C × 10 min, then 40 cycles of 95°C × 15 s, 60°C × 1 min. A melting temperature-determining dissociation step was performed at 95°C × 15 s, 60°C × 15 s, and 95°C × 15 s at the end of the amplification phase. The GAPDH gene was the internal control for the qRT-PCR experiments. Negative controls included reactions without the cDNA template or cDNA reaction products without the reverse transcriptase. Specificity of amplification was confirmed by the melting curve with one peak in PCR reactions.