Computational protocol: Non‐esterified fatty acid‐associated ability of follicular fluid to support porcine oocyte maturation and development

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Protocol publication

[…] The mitochondrial (mt)DNA copy number in the cumulus cells was obtained by dividing the mtDNA copy number by the cell number of the samples and the mtDNA copy number also was determined in individual oocytes. The mtDNA number in the oocytes and cumulus cells was determined by real‐time polymerase chain reaction (RT‐PCR). After IVM, the oocytes were denuded from the cumulus cells, after which individual oocytes were transferred to a PCR tube and the cumulus cell suspensions were centrifuged to obtained cellular pellets. The DNA was extracted from individual oocytes or cumulus cell pellets by using an extraction buffer (20 m mol L−1 Tris‐HCl, 0.9% Nonidet‐40 and Tween 20 and 0.4 mg/mL proteinase K) and by heating at 55°C for 30 min, followed by 98°C for 5 min. The PCR was performed with a Rotor‐Gene 6500 real‐time rotary analyzer (Qiagen GmbH, Hilden, Germany) with a primer that was set to target the mtDNA sequence and the one copy gene and Ssofast‐TM EvaGreen Supermix (Bio‐Rad, Hercules, CA, USA). The primers that were used for the mitochondrial genome and one copy gene are listed in Table . They were designed by using Primer3Plus (http://sourceforge.net/projects/primer3/) and the National Center for Biotechnology Information database (porcine mitochondrion gene, NC_000845.1, and GCG glucagon, NC_010457). The PCR was performed with an initial denaturation at 95°C for 1 minute, followed by 40 cycles at 98°C for five‐seconds and 60°C for 10 seconds. A standard curve was generated for each run by using 10‐fold serial dilutions that represented the copy number of the external standard. The external standard was the PCR product of the corresponding gene that was cloned into a vector by using the Zero Blunt TOPO PCR cloning kit (Invitrogen, Carlsbad, CA, USA) and the PCR product was sequenced for confirmation before use. The amplification efficiencies of all the trial runs were >1.9. [...] All the data were analyzed by using an ANOVA, followed by a post hoc Tukey's test. The percentages were arcsine‐transformed before the analysis. The correlation between the FF NEFA content and the FF concentration was evaluated by a two‐way ANOVA. The analysis was conducted by using IBM SPSS Statistics for Windows software (v. 21; IBM Corporation, Armonk, NY, USA). P‐values that were <.05 were considered to be statistically significant. […]

Pipeline specifications

Software tools Primer3, SPSS
Applications Miscellaneous, qPCR
Chemicals Adenosine Triphosphate