Microarray Studies
For microarray studies left and right testes were individually harvested from control and Tmem203 null mice at 24 weeks of age. At necropsy the left and right testis were individually weighed and recorded along with individual body weights and brain weights in order to assess the combined left and right testes weights relative to body and brain weights. Total RNA was extracted from the testes using RNAasy miniprep kit (Qiagen). Transcriptional profile of each of the nine samples (4 wild type testes and 5 null mice testes) was probed by using Affymetrix Mouse Genome 430 2.0 GeneChips. The raw data obtained after scanning the arrays were assessed for quality by array QualityMetrics [32], a Bioconductor (http://bioconductor.org/) package for quality assessment of microarrays. Robust Multichip Average (RMA) normalization consisting of three steps: a background adjustment, quantile normalization and finally summarization, was applied before obtaining model-based gene expression indices, also known as signal values [33]. Principal Component Analysis was applied on the RMA normalized data to assess for intra group versus inter group variability. All samples were retained for further downstream analyses. All profiling data has been deposited in the Geo Database (http://www.ncbi.nlm.nih.gov/geo/) with the geo accession number GSE66791.
To identify differentially expressed genes between wild type and null testes samples, LIMMA (Linear Models for Microarray Analysis), a Bioconductor (http://bioconductor.org/) package for assessing differentially expressed genes from microarrays using linear models and empiral Bayes methods, was used [34]. All the analyses were done at probeset level. A probeset was retained for further analyses if it showed a 1.5 fold change in the wild type vs null comparison at p < 0.05 (after adjustments for false discovery rate). Additional filters for detection (signal ≥ 32 in at least one chip for a given probeset) and probeset quality (probesets mapping uniquely to only one gene) were applied to account for high quality probesets.
To identify differentially regulated pathways in the null vs wild type testes, pathways analysis (at gene level) was performed. Only one probe set per gene was selected based on the most significant differential p-value. To this end, pathway was performed using IPA (Ingenuity Systems, www.ingenuity.com).