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 8x8 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 (R(2)>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.