The quality of RNA isolation from frozen granulosa cells

Background: Granulosa cells express some important genes for oocyte development and fertilization which may give assessment biochemically to determine the optimal embryos. The stabilization of RNA from frozen cells would affect the quality of mRNA isolation because it is a prerequisite for any gene expression analysis. The purity and concentration from RNA isolation are the two key variable for successful RT-PCR experiment. Methods: The study was done on 28 patients who undergo IVF cycle in Yasmin IVF Clinic, Ciptomangunkusumo Hospital, Jakarta and Daya Medika Kedoya Clinic. The study was approved by the medical ethics committee of Faculty of Medicine, Universitas Indonesia. Informed consent was obtained from all the study participants. Frozen granulosa cells were collected during oocyte retrieval were preserved using RNAlater® Ambion and stored at -80° C, while fresh granulosa cells directly go trough isolation. RNA isolation was conducted using QIAamp RNA Blood Mini Kit QIAGEN. Descriptive study was conducted based on purity and concentration as the result of RNA isolation. Gene expression analysis also conducted with Bax/Bcl2 ratio between two groups. Result: This study result showed the mean concentration of RNA isolation from frozen graulosa cells is 21.78±13.09 ng/μl, from the highest (47.90 ng/μl) to the lowest (8.50 ng/μl). The mean purity of RNA isolation from frozen graulosa cells is 1.85±0.37. In comparison, frozen granulosa cells have lower mean concentration of RNA isolation than from fresh RNA isolated granulosa cells (250.81±229.67 ng/μl). However, for RT-PCR result, there is no significant difference of Bax/Bcl2 ratio between two groups. Conclusion: These findings suggested that RNA quality of frozen granulosa cells is reliable for gene expression analysis. Nevertheless, further studies about the comparison between the RNA quality of frozen granulosa cells and fresh isolated granulosa cells are needed.