Fast start dna green master kit

RT-quantitative polymerase chain reaction (qPCR) was carried out in a LightCycler^® 96 Real-Time PCR System (Roche). The amplification of products was examined through intercalation of the fluorescent dye SYBR^® Green (FastStart DNA Green Master Kit; Roche). The reaction mixture contained 10 μL 2× SYBR^® Green Master Mix, 0.5 μM of each reverse and forward primer, 2.5 ng cDNA, and the appropriate amount of nuclease-free water to bring the final volume to 20 μL. All samples were run in triplicate. A nontemplate control and 4 standards (1:1, 1:10, 1:100, and 1:1,000) were also included. The PCR conditions were determined separately for each target gene according to the melting and annealing temperatures of the primers. Each parameter included a preincubation step for 10 min at 95°C followed by 45 cycles of 3 amplification and melting steps. A melting curve analysis was performed to verify primer specificity. For the quantitation of RT-qPCR results, the ΔΔC_t method was employed (2^−ΔΔCt).

RT-qPCR was carried out in ‘’Light Cycler 96 (Roche)’’. Amplification of products were detected via intercalation of the fluorescent dye SYBR green (Fast Start DNA Green Master Kit, Roche). Briefly, total volume of reaction mix was 20 µl; containing 10 µl ‘’SYBR Green Master Mix (2X)’’, 0.5 µM of reverse and forward primers, 2.5 ng cDNA and appropriate amount of nuclease free water. All samples were run as triplicates in each run including a non-template control and four standards (1:1, 1:10, 1:100, 1:1000). The PCR parameters were determined separately for each target according to melting and annealing temperatures of primers. Each parameter included a pre-incubation step for 10 minutes at 95°C and followed by 45 cycles of three amplification and melting step. Melting curve analysis was performed to verify specificity. For quantitation of RT-qPCR results, ΔΔCt method was used (2-^ΔΔCt).

A Fast Start DNA Green Master Kit (Roche Diagnostics) was used for the real-time reverse transcription quantitative polymerase chain reactions (RT-qPCR). The analysis was performed according to the scientific principles (17 (link), 18 (link)) and the manufacturers’ instructions. Briefly, the total volume of reaction mix was 20 µL, containing 10 μL Master Mix, 10 mM of reverse and forward primers, 25 ng template cDNA and the appropriate amount of RNAse free distilled water. All samples were run as triplicates in each run, including a non-template control and four standards (1:1, 1:10, 1:100, 1:1000). The real-time RT-qPCR parameters were determined separately for each target according to the melting and annealing temperatures of the primers. Each parameter included a pre-incubation step for 10 min at 95°C, followed by 45 cycles of three amplification and melting steps. Melting curve analysis was performed to verify specificity. Absolute quantification analysis was performed using a Light Cycler 96 (Roche Diagnostics). For quantitation of real-time RT-qPCR results, the ΔΔC_t method was used. The gene expression results were represented as ‘Target/GAPDH Fold Change’.