Mouse gene 2.0 st genechip

Total RNA samples from the corpus and the cauda epididymal regions of three controls and three Dicer1 cKO mice were used to determine the transcript signature found in these regions. Microarray analyses were performed by the CHUQ Gene Expression Platform (Quebec, Canada). Samples were hybridized to the GeneChip Mouse Gene 2.0 ST (Affymetrix). This array encompasses 35,240 transcript probe-sets, from three transcript data sources (i.e. RefSeq, Ensembl and lncRNA db). As described above, microarrays were scanned using the Affymetrix GeneChip Scanner 3000 7G and Affymetrix GeneChip Command Console software (Affymetrix, Santa Clara, CA), to produce the intensity files. The image data were analyzed with Expression Console Software for quality control (Affymetrix) (S2 Fig) and extracted with the GeneChip Operating Software (GCOS v 1.4; Affymetrix). CEL files were imported and analyzed with Partek Genomics Suite 6.5 software (Partek Incorporated). All gene expression microarray data have been deposited in the Gene Expression Omnibus (GEO) repository database for public access (#GSE77139).

mRNA isolation and quality controls were performed as described in References [31 (link),32 (link)]. The mRNA samples isolated from the three independent cell culture replicates were mixed in equal mRNA quantities. The procedures for cDNA synthesis and labelling were carried out according to the Ambion WT Expression Kit (Life Technologies, Darmstadt, Germany) using 500 ng of total RNA as a starting material, as described in Reference [33 (link)]. Target DNA fragmentation, hybridization on Affymetrix GeneChip Mouse Gene 2.0 ST microarrays, array washing, staining, and scanning were performed as described in Reference [34 (link)]. Scans of the microarrays were converted into CEL files using the scanner software, and were then processed in Affymetrix Expression Console (build 1.4.1.46) using the RMA method. Fold change threshold was set to 1.5×. Probesets with no associated Gene Symbol were excluded from the analyses. The raw data have been deposited to the Gene Expression Omnibus [35 (link)] under accession code GSE96899.

Total RNA was isolated using the RNeasy spin column kit (Qiagen) and quantified using a BioSpectrometer (Eppendorf). One hundred nanograms of RNA was used as input for the GeneChip® PrimeView™ (3′ IVT human array) and GeneChip™ Mouse Gene 2.0 ST (mouse exon array) microarrays (Affymetrix). Synthesis, labeling, and purification of biotinylated complementary RNA and sense-stranded DNA targets were carried out according to manufacturers’ instructions using the GeneChip™ 3′ IVT PLUS Reagent Kit and GeneChip™ WT PLUS Reagent Kit, respectively. Hybridization was performed using a GeneChip Hybridization Oven 640 overnight at 45 °C. Microarray washing and staining was performed on a GeneChip Fluidics Station 450 and scanning on a GeneChip Scanner 3000 7G, commanded by the Affymetrix GeneChip Command Console software. Probe-level analysis including background subtraction and quantile normalization took place with the Robust Multi Array Average Algorithm using the Affymetrix Expression Console Software 1.3. DEGs (p < 0.05 and fold change > 1.5 for the GeneChip™ Mouse Gene 2.0 ST exon array and p < 0.01 and fold change > 1.5 for the GeneChip® PrimeView™) were determined using the Transcriptome Analysis Console v3.0. Raw and processed Affymetrix data have been deposited in the Gene Expression Omnibus repository under accession number GSE120127.