Quantitative Analysis of Real-Time PCR Expression Production by REST and 2~((-ΔΔCT))

Objective To develop a simple and accurate method for the quantification of real-time RT-PCR gene products.Method Standard curves of POLK and GAPDH genes were made from 5-fold serially diluted cDNA samples from the normal 16HBE cells.The levels of POLK mRNA in the malignant transformed 16HBE cells and human lung cancer cells(H1299) treated with various concentrations of BPDE were determined.GAPDH gene was used as the reference gene.The relative expression levels were analyzed by REST and 2(-ΔΔCT) method.Result A good linear relationship(Rsq 0.997) was obtained from the standard curves.The efficiency of amplification of POLK and GAPDH genes was 117.5% and 103.5%,respectively.The expression ratios calculated by using 2(-ΔΔCT) method were higher than the rations obtained from the REST.Conclusion Real-time RT-PCR in combination with REST is a simple and accurate method for relative quantification of gene expression.