Comparison of real-time polymerase chain reaction and end-point polymerase chain reaction for the analysis of gene expression in preimplantation embryos

Abstract

The aim of the present study was to compare real-time polymerase chain reaction (PCR) and end-point PCR with respect to their suitability for the analysis of gene expression in samples in which the number of cells is limited; for example, in studies of preimplantation embryonic development and to determine the variability of the real-time reverse transcription-PCR assay. The sensitivity, dynamic range and precision of both PCR systems were compared using a single mouse liver cDNA standard. The real-time system was 100-fold more sensitive than the end-point system and had a dynamic range of more than four orders of magnitude. The linear range for end-point PCR extended for two orders of magnitude using a fixed end-point of 31 cycles. The percentage standard error of the mean based on 30 replicates was 0.14% of the threshold cycle (Ct) value for the real-time system and 6.8% for the end-point fluorescence intensity. The coefficients of variation (CV) for reverse transcription combined with real-time analysis and the complete gene expression protocol consisting of mRNA isolation, reverse transcription and real-time PCR analysis were 0.6% and 1.4% of the Ct values, respectively. The present paper details, for the first time, measurement of the biological variation of individual mammalian oocytes. The CV was 1.8% of the Ct value for expression analysis of six bovine oocytes. The results are discussed in relation to the analysis of gene expression in preimplantation embryo development.

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