Comparison of real-time polymerase chain reaction and end-point polymerase chain reaction for the analysis of gene expression in preimplantation embryos
Árpád Baji Gál A D , Joseph Wallace Carnwath B , Andras Dinnyes A C , Doris Herrmann B , Heiner Niemann B and Christine Wrenzycki BA Agricultural Biotechnology Center, Institute of Animal Biology, Szent-Györgyi Albert u. 4, 2100 Gödöllő, Hungary.
B Institute for Animal Science, Department of Biotechnology, Höltystr. 10, 31535 Neustadt, Germany.
C Hungarian Academy of Sciences and Szent István University, Páter K. u. 1, 2103 Gödöllő, Hungary.
D Corresponding author. Email: baji@abc.hu
Reproduction, Fertility and Development 18(3) 365-371 https://doi.org/10.1071/RD05012
Submitted: 28 January 2005 Accepted: 25 November 2005 Published: 27 January 2006
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.
Acknowledgments
The authors thank Martina Waßmann for assistance in running the LightCycler. This research was supported financially by the Bilateral Scientific and Technological Collaboration Agreement (TET) between Hungary and Germany (TET #D-6/01) and by the National Office of Research and Technology (NKTH; #BIO-00017/2002).
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