Analysis of gene expression in single oocytes and embryos by real-time rapid cycle fluorescence monitored RT-PCR

Abstract

Rapid cycle DNA amplification is a refinement of the polymerase chain reaction (PCR) method that permits increased product specificity while reducing amplification time by an order of magnitude. Combined with the use of micro volume capillaries, minute samples can be examined by this technique. Thus, this approach is ideally suited to the analysis of gene expression in individual cells. As the current understanding of early developmental processes is still rudimentary, further characterization of transcription in single oocytes and embryos may provide additional insight into the molecular mechanisms directing these events. In this study, we examined the suitability of fluorescence monitored reverse transcription (RT)-PCR for the study of gene expression during oogenesis and embryogenesis using transcripts of the housekeeping gene, beta-actin, as an experimental model. Product accumulation was monitored by either the double-stranded DNA dye SYBR Green I or sequence-dependent hybridization of reporter molecules called molecular beacons. Dyes bind generically and are economical to use. However, both specific and non-specific products are labelled. Hybridization probes permit very specific and sensitive target recognition but they can be costly to manufacture. Once molecular markers indicative of optimal development are identified, this technology could be used in a clinical in-vitro fertilization laboratory as a diagnostic tool.

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