title: OPTIMISATION OF TOTAL RNA EXTRACTION FROM BOVINE OOCYTES AND EMBRYOS FOR GENE EXPRESSION STUDIES AND EFFECTS OF CRYOPROTECTANTS ON TOTAL RNA EXTRACTION.
abstract: Gene expression is required for understanding bovine oocytes meiotic maturation as well as the potential of embryonic development. In the present study a standardized reagent protocol for total RNA extraction was designed for bovine oocytes and embryos, which is considered specific and less expensive. For such purpose oocytes (n = 795) recovered from about 80 ovaries were divided in three groups: Group 1 modified Trizol (MTP, n = 355); Group 2 Guanidinium thiocyanate protocol (GNTC, n = 140) and Group 3 Commercial Kit protocol (CKP, n = 60). Oocytes belonging to group 1 (n = 100) and 3 (n = 20) were subjected to vitrification using two cryoprotectants 1,2 propandiol (PROH) or Dimethylsulfoxide (DMSO). The 240 remaining oocytes were divided into 3 groups in which 100 were used, in fresh, for in vitro fertilization, and 140 oocytes were vitrified using PROH (n = 70) and DMSO (n = 70) as cryoprotectants, being then fertilized in vitro after thawing. Embryos were used nine days after fertilization. Gene amplification (SDHA, (GAPDH and DNMT1) was performed in oocytes, and gene quantification (DNMT1) in in vitro produced embryos at the stage of blastocyst (n = 10). Efficiency of the extraction was further compared. The purity of all samples to different protocols ranged from 1.10 to 1.25 for GNTC protocol; from 2.05 to 2.63 for the CKP and from 1.50 to 2.11 for the developed MTP, being the last one nearest to the expected purity levels for RNA samples (1.7 to 2.0). On average, for 30 fresh oocytes, from spectrophotometer readings, total RNA concentration was 127.8 ± 9.3 ng μl(-1) for MTP, against 46.4 ± 9.5 ng μl(-1) from CKP and 476 ± 12.9 ng μl(-1) for GNTC protocol. Using the MTP to evaluate RNA in 30 vitrified/thawed oocytes, resulted in a total RNA concentration of 61.3 ± 3.3 ng μl(-1) and 40.0 μ 12.4 ng μ(-1), respectively for DMSO and PROH. Regarding total RNA concentration and purity, in blastocyst stage, more purity was observed in DMSO as compared to PROH (1.8 vs 1.2) (p < 0.05). Better results were also observed on the MTP for gene amplification when compared with the other protocols. 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.
authors.forename: K C
authors.initials: KC
authors.lastname: Pavani
authors.forename: E E
authors.initials: EE
authors.lastname: Baron
authors.forename: M
authors.initials: M
authors.lastname: Faheem
authors.forename: A
authors.initials: A
authors.lastname: Chaveiro
authors.forename: F Moreira
authors.initials: FM
authors.lastname: Da Silva
mesh_terms: D000818:Animals; D002417:Cattle; D003451:Cryoprotective Agents / Q000494:pharmacology; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005307:Fertilization in Vitro; D015870:Gene Expression; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D006146:Guanidines / Q000737:chemistry*; D008297:Male; D009865:Oocytes / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010636:Phenols / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D019946:Propylene Glycol / Q000494:pharmacology; D012313:RNA / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013385:Succinate Dehydrogenase / Q000235:genetics; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; D006146:Guanidines; D010636:Phenols; C411644:trizol; D012313:RNA; D019946:Propylene Glycol; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D013385:Succinate Dehydrogenase; D004248:DNA (Cytosine-5-)-Methyltransferases; D004121:Dimethyl Sulfoxide