Validation of universal conditions for duplex quantitative reverse transcription polymerase chain reaction assays

Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used for measuring mRNA in biological materials. Multiplex qRT-PCR provides advantages for gene expression analysis by reducing sample requirements, saving time, and lowering experimental cost. However, there are currently no universal qRT-PCR experimental conditions validated as applicable to a large set of genes. We report here on the standardized condition for two-color real-time qRT-PCR with the Quantitect Multiplex PCR kit. We first verified lack of interferential effects of gene abundance on the efficiency of PCR amplification by an 8 × 8 checkerboard validation method, in which combinations of the plasmids encoding either fibronectin1 or cyclophilin mixed at 64 different ratios were amplified with the Quantitect Multiplex PCR kit. Then, a duplex analysis for 69 genes was performed to verify the universality of the reaction condition. The results were consistent with corresponding data obtained from the singleplex format, and their intra- and interassay coefficients of variance were sufficient for performing reliable quantitative analysis. This duplex format was also applicable to samples from animal experiments, with a good correlation between singleplex and duplex-assay (R2 > 0.92) observed. This duplex assay system is ready for use in high-throughput gene expression analysis without any gene-pair compatibility restrictions limiting its use.