In addition, no specific bands were detected in reactions containing the tRNA carrier alone. To confirm the integrity of the RNA templates and the RT-PCR protocol, p-actin was examined in all samples. Furthermore, zona pellucida 3 (ZP3) and GDF9 were amplified as oocyte-specific markers, while the ovary-specific transcript of the human P450arom was used to ensure the absence of granulosa cell mRNA in the oocyte RNA extracts. For transcripts that were negative in human oocytes, granulosa cell RNA was used as the positive control, while mouse ovary total RNA was used as a positive control sample for mouse oocytes. Mouse embryos were analyzed as a control for the sensitivity of the PCR for targets that were undetectable in oocytes. Buy Advair Diskus Online
Background, as determined by counting the no-template control lane, was subtracted from each target signal, and the results were grouped as mean ± SEM, collectively or according to the maturation stage of the oocyte. Samples that produced a negative or weak p-actin signal, or showed contamination in reverse transcriptase- or template-deleted controls, were excluded from analysis. Human oocyte samples that produced a strong P450arom signal were considered to include granulosa cell RNA, and data for these were deleted. Experiments in which mouse ovary RNA failed to produce positive signals were also excluded. Statistical differences between maturation groups were determined using Mann-Whitney U test. Differences were considered significant at p < 0.05. For comparison of human oocytes with cumulus cells and with mouse oocytes and embryos, the number of samples in which each target was detected by autoradiography was reported as a percentage of the total number of samples.