Computational protocol: The periovulatory endocrine milieu affects the uterine redox environment in beef cows

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Protocol publication

[…] Subsets of 9 cows per group were selected for transcript quantification analyzes (please see Statistical Analyses section for details). Approximately 30 mg of endometrial tissues were submitted to total RNA extraction, using the RNeasy Mini columns kit (Qiagen, Gaithersburg, MD, USA) according to the manufacturer’s instructions. To complementary DNA synthesis 1 μg total RNA was treated with DNAse I followed by reverse transcription using a High Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA). Step-One Plus (Life Technologies) with SYBR® Green Chemistry was used for the amplification reactions.Primers were designed based on GenBank Ref-Seq mRNA sequences of target genes, using the Primer Express software, version 3.0 (Life Technologies). The specificity of the designed primers was compared by Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov). PCR products of the primers designed were submitted for electrophoresis and sequencing. Details of the primers and probes are provided in Table .Determination of PCR efficiency and Cq (quantification cycle) values per sample were performed with LinRegPCR software (http://linregpcr.nl/) as described by Ramakers et al. []. For data normalization, Genorm software was used as described by Vandesompele et al. []. Three constitutive genes – cyclophilin (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-actin (ACTB) – were used as inputs. The normalization factor generated by Genorm was based on the geometric mean of the most stable genes (GAPDH and ACTB). [...] The experimental model was used as a paradigm for lower (SF-SCL) or greater (LF-LCL) receptivity and fertility. Adherence to each of the paradigms was measured in each animal after joint evaluation of specific ovarian and endocrine variables, specifically, concentration of P4 at D6, P4 at D6/P4 at D2 ratio, CL size at D7, CL weight, follicle size at D-2, D-1 and D0 and pre-ovulatory follicle size. Animals within each group were ranked according to responses to each variable. The nine top ranked animals of the LF-LCL group and the lowest ranked animals of the SF-SCL group were chosen for biochemical and transcript analysis. Raw data were checked to determine the normality of the residuals by the Shapiro-Wilk test, and Levene’s test was used to check for homogeneity of variances. If necessary, data were transformed by natural logarithms or ranks. Comparisons between the groups were analyzed by one-way ANOVA using the PROC GLM procedure (SAS software, version 9.2). The P4 concentration was analyzed by split-plot ANOVA, considering the effects of group, day, and their interaction using the PROC MIXED procedure (SAS, Version 9.2; SAS Institute Inc., Cary, NC, USA). The data from the biochemical analyses were compared between the groups by one-way ANOVA (STATISTICA 4.5, StateSoft, Inc. 1993, Tulsa, OK, USA). […]

Pipeline specifications

Software tools Primer Express, BLASTN, LinRegPCR, Statistica
Applications Miscellaneous, qPCR
Organisms Bos taurus
Chemicals Estradiol, Glutathione, Gonadotropin-Releasing Hormone, Malondialdehyde, Progesterone, Steroids, Superoxides