RNA ISOLATION AND DETECTION OF CELLULAR RNA QUANTITY OF SPERMATOZOA AND EMBRYOS PRIOR TO GENE EXPRESSION ANALYSES

DOI: 10.26650/IUITFD.416666 Objective: Biological samples that are analyzed in reproductive biology are generally rare, difficult to obtain, and a nonreplicable group of cells. Furthermore, investigating low numbers of cells requires modifications to the routine methods used in genetic analyses. The aim of our study was to improve RNA isolation methods for obtaining a sufficient amount of total RNA from spermatozoa and embryo samples for downstream gene expression analyses. Materials and Methods: Excess spermatozoa samples (that had been prepared in the scope of assisted reproduction treatment) were purified of any contaminant cells and then frozen. Preimplantation stage embryos that had not been selected for embryo transfer were frozen in a phosphate-buffered saline–polyvinyl alcohol (PBS–PVA) solution. Modifications were done to obtain the optimal amount of total RNA from the spermatozoa and embryos, and subsequently both the quality and the quantity of the total RNA samples were assessed. Because of the selective degradation of 28S RNA samples in the spermatozoa, the total RNA quality was evaluated by gene amplification using quantitative real-time polymerase chain reaction (qRT-PCR) in addition to Bioanalyzer analysis. Results: Following the modifications to the isolation technique, a sufficient quality and quantity of total RNA were obtained from the spermatozoa and embryo samples, which could be used in downstream gene expression analyses. Furthermore, the amount of cellular total RNAs was consistent to that reported in previous studies. Conclusion: Results obtained from these experiments - conducted as a preliminary work for further transcript profiling studies - indicate that the modifications used in the RNA isolation techniques were effective