210 COMPARISON OF REAL-TIME PCR AND END-POINT PCR FOR ANALYSIS OF GENE EXPRESSION IN PREIMPLANTATION EMBRYOS

Abstract

The purpose of this study was to compare real-time 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 pre-implantation embryonic development. The real-time instrument was a LightCycler^® from Roche Diagnostics (Budaors, Hungary) which is a capillary-based PCR system. Primers for histone H2A (housekeeping gene) were used for all PCR reactions. The end-point PCR system included an MJ Research PT-100 thermocycler, agarose gel electrophoresis, ethidium bromide staining, and image acquisition with a 12-bit CCD camera and densitometry. The sensitivity, dynamic range, and standard error of both PCR systems were compared using a single stock solution of cDNA. The more precise real-time PCR system was then used to determine the precision of a protocol for reverse transcription and the precision of a complete gene expression protocol including mRNA purification and reverse transcription. The real-time system was 100 times 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 was extended for two orders of magnitude using a fixed end-point of 31 cycles. The standard error of the mean based on 30 replicates was 0.14% for the real-time system and 6.8% for the end-point system. The standard deviations for reverse transcription combined with real-time analysis and for the complete gene expression protocol were 0.6% and 1.4%, respectively. The t standard deviation was 1.8% for expression analysis of 6 bovine oocytes. In conclusion, real-time PCR system has advantages in sensitivity, dynamic range, and precision of measurement. New research areas which involve subtle changes in expression reprogramming or the analysis of low copy number transcripts (even from single cells and embryos) clearly benefit from the advent of real-time PCR analysis. However, when genes with high transcription levels are analyzed, the amount of cDNA taken from the reverse transcription reaction can be adjusted to lie within the operating range of end-point PCR. Pooling embryos is a valuable approach for both methods when the goal is to determine the behavior of the average embryo rather than variation between embryos. In many cases, the magnitude of biologically significant expression changes is so great that the higher levels of precision afforded by real-time PCR are not essential for the analysis.

This work was funded 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).