Quantification of mRNA in single oocytes and embryos by real-time rapid cycle fluorescence monitored RT-PCR

Deciphering the complex series of regulatory events that occur during early development depends partly on the ability to accurately quantify stage-specific mRNA species. However, the paucity of biological material coupled with the lack of sensitivity and/or reproducibility of the currently available quantitative methods had been severe limitations on single cell analysis. Rapid cycle DNA amplification is a highly sensitive technique for amplification of specific DNA sequences. With the addition of fluorescence probes, it is possible to monitor the log-linear phase of amplification during which the most useful quantitative data is obtained. Unknown concentrations are extrapolated from standards co-amplified producing a standard curve. Furthermore, micro volume capabilities allow for the analysis of minute samples. Consequently, this approach is ideally suited to the needs of the clinical IVF laboratory. Rapid fluorescence monitored cycling was used to examine expression levels of the housekeeping genes beta-actin and hypoxanthine guanine phosphorlbosyltransferase in individual murine/human oocytes and/or embryos. Results obtained compared favourably with those attained by others and followed the predicted temporal patterns of expression. Once informative reproductive molecular markers are identified by micro-array analysis, minimally invasive techniques can be developed to biopsy cytoplasm and/or polar bodies for clinical evaluation using rapid fluorescence monitored reverse transcription-polymerase chain reaction methods.