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

Rapid cycle DNA amplification is a refinement of the polymerase chain react ion (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 ind ividual cells. As the current understanding of early developmental processe s is still rudimentary, further characterization of transcription in single oocytes and embryos may provide additional insight into the molecular mech anisms directing these events. In this study, we examined the suitability o f fluorescence monitored reverse transcription (RT)-PCR for the study of ge ne expression during oogenesis and embryogenesis using transcripts of the h ousekeeping gene, beta-actin, as an experimental model. Product accumulatio n was monitored by either the double-stranded DNA dye SYBR Green I or seque nce-dependent hybridization of reporter molecules called molecular beacons. Dyes bind generically and are economical to use. However, both specific an d non-specific products are labelled. Hybridization probes permit very spec ific 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 laborato ry as a diagnostic tool.