Faststart dna master sybr green kit

In the study, two positive controls were used, P3HR1 and EBV + AKATA mRNA. To ensure the accuracy and reliability of the results, actin was used as a standard control. For the RT-PCR process, reverse transcription was performed, followed by quantitative PCR using the FastStart DNA Master SYBR green kit (Roche Diagnostics, Meylan, France) in a final volume of 20 µL. The primer sequences used in the study are provided in Table 1.
To determine the mRNA concentration, a calibration curve was utilized. The size of the amplified products was verified by gel electrophoresis using molecular weight markers during the initial PCR setting adjustment. Amplification specificity was checked using light cycler melting-curve analysis once the PCR product size was confirmed. The Light Cycler quantification software 4.1 quantified the transcripts in samples, and the data were calibrated from repeated dilutions of purified PCR products containing known amounts of cDNA molecules from each gene (Roche Diagnostics). The ratio of LMP1 mRNA to actin mRNA was calculated to determine the relative abundance of each mRNA. This experiment was replicated three times.
$$\mathrm{Normalized} \mathrm{ratio}={\left(\frac{\mathrm{conc}. \mathrm{target}}{\mathrm{conc}. \mathrm{reference}}\right)}_{\mathrm{sample}}:{\left(\frac{\mathrm{conc}. \mathrm{target}}{\mathrm{conc}. \mathrm{reference}}\right)}_{\mathrm{calibrator}}$$

RNA was reverse transcribed using the Ready-To-Go You-Prime First-Strand Beads (GE Healthcare, Fairfield, CT) and Random Primers (Invitrogen). Primers were designed with Primer 3 software (Whitehead Institute for Biomedical Research, Boston, MA). Quantitative Real-Time PCR analysis was carried out on the capillary-based Light Cycler (Roche, Basel, Switzerland) using the FAST Start DNAMaster Sybr Green Kit (Roche). Relative expression of cDNA of the target gene in comparison to a reference gene was calculated using a mathematical model proposed by Pfaffl [13] (link). Samples were analysed in duplicate and averaged. Calculated cDNA amounts of the target genes were normalized to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All data are represented as ratio of the target gene/GAPDH. Primers are shown in table S2 in file S1.

Total RNA was extracted from the cell lines using TRIzol (Invitrogen, USA) and was reverse transcribed using the First Strand cDNA synthesis kit (Takara, Japan). The products were used as templates for the PCR assays using a FastStart DNA Master SYBR Green Kit (Roche, Germany) in the Light Cycler 96 sequence detection system. PCR was carried out following the manufacturer's instructions. All reactions were performed in triplicate, and β-actin was used for normalization. The 2^-ΔΔCt method was used to assess the relative gene expression. The PCR primers are shown in Table 1.