Computational protocol: Peg3 Mutational Effects on Reproduction and Placenta-Specific Gene Families

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Protocol publication

[…] A litter of 14.5-dpc fetuses were harvested from a timed mating between a male heterozygote (−m/+) and wild-type female littermate. Three tissues were obtained from each fetus: embryo head, placenta and amnionic sac. The DNA from amnionic sac was used for determining the genotype and gender of each fetus. Embryo heads and placenta were used for isolating total RNA. Total 16 samples were used for this series of genome-wide expression analyses: 8 embryo heads and placenta representing two biological replicates of each of the four following combinations: male wild-type, male heterozygote, female wild-type, and female heterozygote. The isolated total RNA from each sample was further treated with DNAse I and later purified with columns to remove any genomic contaminations (Qiagen, RNeasy Mini-kit). Each total RNA (2 µg) was used for generating labeled cRNA for hybridization on a mouse expression analysis platform (Illumina, Microarray-Mouse Ref-8 v2 expression beadchip). In brief, each cRNA sample (750 ng) was hybridized onto the expression bead chips at 58°C overnight. Chips were scanned with the Illumina Bead Array Reader (Factor = 1, PMT 520, Filter = 100%), and the numeric results were extracted with GenomeStudio using the Gene Expression Module v.1.0.6. Raw data were background-subtracted and normalized using the quantile normalization method (lumi software package) ,. Normalized data were filtered for genes with significant expression levels compared to negative control beads. Selection for differentially expressed genes was performed on the basis of arbitrary thresholds for fold changes plus statistical significance according to the Illumina t-test error model (limma software) . The mRNA array data in MIAME compliant has been submitted to the NCBI Gene Expression Omnibus (GEO) database (Accession No. GSE50818). Pathway analyses were also performed with the EGAN (Explorative Gene Association Networks) package with the obtained gene sets with up- and down-regulation . […]

Pipeline specifications

Software tools GenomeStudio, lumi, limma
Databases GEO
Application Gene expression microarray analysis
Organisms Mus musculus, Homo sapiens