Abstract: OBJECTIVE: To identify a proper qPCR method for examining expression of methylation-related genes in mouse early embryonic cells. METHODSNormal qPCR,isothermal pre-amplification-based qPCR,and PCR pre-amplification-based qPCR methods were applied in quantitative assay of methylation-related genes (TET1,TET2,TET3 and DNMT3A) in mouse early embryonic cells. RESULTS: The sensitivity of these three methods decreased from isothermal pre-amplification-based qPCR method,PCR pre-amplification-based qPCR method to normal qPCR method. The former two methods detected expression of methylation-related genes in a few mouse embryonic cells within a proper PCR cycle number but the PCR pre-amplification-based qPCR method has lower cost than isothermal pre-amplification-based qPCR method. Therefore,we used PCR pre-amplification-based qPCR method to examine methylation-related genes in mouse oocytes and early embryonic cells after 22 h fertilization. The results showed that the expression level of TET1 mRNA was very low and not detectable,the expression of TET2 decreased,and the expression of TET3 and DNMT3A significantly increased (P<0.01) in this process,which was consistent with reported results. CONCLUSION: The sensitivity of isothermal pre-amplification-based qPCR method was the highest among the three qPCR methods. However,the PCR pre-amplification-based qPCR method is the most suitable one for examining multi-gene expression in a few cells in daily practice due to the simple procedure relatively high sensitivity and low expenditure.

Key words: pre-amplification, real time quantitative PCR, mouse, early embryonic cell, methylation-related genes