Computational protocol: Long-Term and Transgenerational Effects of Stress Experienced during Different Life Phases in Chickens (Gallus gallus)

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[…] After culling, the birds were decapitated and their brains were removed, dissected and frozen in liquid nitrogen within 15 min. From the brains, we dissected the ventral half of the mid-brain, mainly consisting of thalamus and hypothalamus []. Samples from parents and offspring were treated and analysed with the same methods, according to the following procedure:Brain samples were homogenized with 1 ml TRI-reagents (Ambion) in Lyzing matrix D tubes containing ceramic beads (MP Biomedicals) using a FastPrep® -24 homogenization system. The rest of the extraction was performed according to the TRI-reagent manufacturer’s protocol, with the modification that 0.25 ml isopropanol and 0.25 ml RNA-precipitation solution (1.2 M NaCl, 0.8 M disodium citrate) was added to the samples during extraction. RNA quantity and quality was measured using a NanoDrop® ND-2000c (Thermo Scientific) and a Bioanalyzer® instrument (Agilent Technologies). All samples were individually treated with DNase I (Thermo Scientific). Then first-strand cDNA was synthesized using Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Scientific), followed by second strand cDNA synthesis, phenol/chloroform extraction and precipitation. Samples were pooled (see below), labeled and hybridized to NimbleGen 12 x 135k custom gene expression arrays (Roche NimbleGen) and scanned using a NimbleGen hybridization station and scanner. For gene expression data, the dscDNA samples from the brains were pooled in groups of three within generation, treatment and sex before labeling and hybridization. In this way, we had one microarray with a pool of three individuals for each generation, sex and treatment group, altogether 48 birds. The microarray data can be accessed at Annotare ( under accession E-MTAB-3623. [...] Statistical analyses were performed in SPSS version 22 or Statistica 12 (StatSoft, Tulsa, OK, USA), and in R (gene expression). One-way ANOVA was used to determine between group differences in egg mass laid by stress treated females. The undisturbed behaviour was analyzed using a factorial ANOVA with sex and treatment in the model. Open field behavioural data for offspring and body mass data for all treatment groups and their offspring were analysed by two-way ANOVA with treatment and sex as variables. Plasma CORT values from the restraint test were analysed using a mixed repeated-measure ANOVA with treatment and sex as between-subject factors and time as within-subject factor. A repeated measures ANOVA was used for analyzing the stress recovery/novel object test. The between treatment group difference in duration of tonic immobility and the emergence test were examined by using Kaplan-Meier survival analysis, with a post hoc χ2-test or Cox’s F-test. The statistical significance level was set at P < 0.05. If 0.05 > P < 0.1, the results were considered a tendency and a post-hoc analysis was performed.Normalization of the arrays was done with the RMA method using the DEVA software (NimbleGen). Gene expression was analyzed using R/Bioconductor ( each stress treatment group, the expression was compared to the controls. The genes were listed according to fold-change, disregarding P-values, and the top 1000 genes on these lists were obtained for each of the stress groups. To analyse pathways affected by the stress treatment, we selected genes overlapping between all treatment groups (i.e. found on the top 1000 list of all three groups) and subjected those to gene ontology analysis. For this purpose, Ensembl gene IDs were matched to the chicken array using the Manteia web tool ( analyse possible transgenerational effects of the stress treatments, we generated the same top 1000 fold-change lists for the F1-birds, comparing expression in the offspring of each treatment group to offspring of the controls. We then selected those genes, which appeared on the top lists of both the parents and the offspring in each treatment group, and calculated the correlation coefficients for each comparison. This analysis shows the extent to which altered gene expression in parents stressed at a certain age is mirrored in the expression profiles of their offspring. […]

Pipeline specifications

Software tools Annotare, SPSS, Statistica
Databases ArrayExpress MANTEIA
Applications Miscellaneous, Gene expression microarray analysis
Organisms Gallus gallus
Diseases Puberty, Delayed
Chemicals Corticosterone