Optimization of a specific messenger RNA extraction protocol for fresh and vitrified bovine oocytes to gene expression studies : Specific mRNA extraction protocol for bovine oocytes.

Sammanfattning: To understand bovine oocytes meiotic maturation, developmental potential, gene expression is required. The gene expression studies in the preimplantation bovine oocytes has been difficult, because the procedures that are being employed for extracting total RNA are not specific for bovine oocytes and so far is not providing the required amount for further procedures. Quantification of genes generally requires large amounts of total RNA in order to overcome the problem of low amount of mRNA present, so a standardized specific protocol is recommended. These days most of the researchers are using commercial Kit protocols without knowing the significance of chemicals and how they are acting on cells. In present project a standardized protocol (modified trizol) was designed for bovine oocytes, which was specific and less expensive. The efficiency of this protocol compared with Pure Link (Kit Protocol), GNTC (Guanidinium thiocyanate) for extraction of total RNA from fresh oocytes, vitrified oocytes with PROH (1,2 propanediol) and DMSO (dimethylsulfoxide) cryoprotectans was much better. The RNA (absorbance 260/280) purity levels of the standardized protocol was ranging (1.50-2.10), whereas for GNTC protocol (1.05-1.36), Pure Link (kit protocol) (2.05-2.7). Amplification of housekeeping genes (SDHA and GAPDH gene) showed the specificity and efficiency of the standardized protocol over other protocols.