Reverse transcriptase pcr kit

In order to evaluate the effects of RNA interference of the psy gene, quantitative real-time RT-PCR was utilized for analysis of all the PSY RNAi strains. The RNA samples for quantitative real-time RT-PCR analysis were collected from cells grown under the same growth conditions. Total RNA was isolated from all the PSY RNAi R. sphaeroides strains and reversely transcribed using a two-step strategy with reverse transcriptase PCR kit (Qiagen, Shanghai, China). The RT-qPCR reaction was carried out in 25 μL reactions containing 12.5 μL of Universal SYBR Master (ROX), 2.0 μL of properly diluted cDNA from 20–30 ng/mL of cDNA for all genes, 0.5 μL of 50× ROX reference dye II (for error correction between wells), and 0.5 ml of each primer at 10 mM and 9 mL of sterile distilled water. GAPDH gene of R. sphaeroides was selected as control for normalizing expression of the samples. All real-time PCR reactions were performed on the LightCycler^® 96 (Roche Diagnostics, US). The fold change of the target cDNA was determined using the value of $2^{-\mathrm{\Delta }\mathrm{\Delta }{\text{C}}_{\text{t}}}\left(-\mathrm{\Delta }\mathrm{\Delta }{\text{C}}_{\text{t}}={\left[{\text{C}}_{\text{t}(\text{target})}-{\text{C}}_{\text{t}(\text{ref})}\right]}_{\text{sample}}-{\left[{\text{C}}_{\text{t}(\text{target})}-{\text{C}}_{\text{t}(\text{ref})}\right]}_{\text{WT}}\right)$ , where Ct represents the threshold cycle [32 (link)]. All the values were determined through triplicate experiments and statistical significance was considered significant at P < 0.05.