Total RNA extraction protocol specific for bovine oocytes and embryos

Título:

Total RNA extraction protocol specific for bovine oocytes and embryos.

Autora:

Krishna Chaitanya Pavani

Descrição:

To extract total RNA, most of the researchers are using commercial Kit protocols without knowing how they act on cells, being a specific protocol recommended for each species. Here a standardized protocol for total RNA extraction was designed for bovine oocytes and embryos, which is considered specific and less expensive. Bovine oocytes (n=770) were divided in three groups: Group 1 modified trizol protocol (MTP n=355); Group 2 Guanidinium thiocyanate protocol (GNTC n=140) and Group 3 Commercial Kit protocol (CKP n=60). Gene amplification (SDHA, GAPDH and DNMT1) was performed in oocytes, and gene quantification (DNMT1) in in vitro produced embryos (n=10) at the blastocyst stage and compared the efficiency of the extraction. On average, for 30 oocytes, total RNA concentration was 127.8±9.3ng µL-1 for MTP against 46.4±9.5ng µL-1 from CKP and 47.6±12.9ng µL-1 for GNTC. Better results were also observed on the MTP for gene amplification, as for the same number of oocytes brighter bands were observed in this protocol when compared with the others. For gene quantification, the proposed protocol quantified DNMT1 gene with PCR efficiency (0.933) after normalization against GAPDH and SDHA. Amplification and quantification of genes proved specificity and efficiency of the MTP over the other protocols.