Figure 1

Real-time PCR quantification of BCL2L12 gene expression in NPC tissue biopsies. (A) Amplification plot of BCL2L12 and GAPDH cDNAs, showing ΔR_n plotted against the cycle number. BCL2L12 mRNA expression was detected by real-time quantitative PCR using SYBR® Green chemistry, with GAPDH as a reference gene. Calculations were made using the comparative C_T (2^−ΔΔCT) method. (B) Dissociation curves of the BCL2L12 amplicon, demonstrating the specificity of primers used for the real-time PCR amplification and quantification of BCL2L12. Neither primer-dimers nor other nonspecific products were observed after melting of the PCR products. (C) Validation of the comparative C_T (2^−ΔΔCT) method to assess the efficiency of amplification of the target gene (BCL2L12) and the internal control (GAPDH). Using reverse transcriptase, cDNA was synthesized from 2 µg total RNA isolated from human HL-60 cells. Serial dilutions of cDNA over a 10⁶-fold range were amplified by real-time PCR using gene-specific primers. The most concentrated sample contained cDNA derived from 100 ng total RNA. BCL2L12 and GAPDH average C_T values were calculated for each cDNA dilution and plotted against log cDNA dilution. All data were fit using least-squares linear regression analysis. The absolute values of the slopes of resulting plots are approximately equal, thus implying similar amplification efficiencies for both genes.