Affymetrix genechip mouse gene 2.0 st array

The Affymetrix GeneChip Mouse Gene 2.0 ST Array (Thermo Fisher) was used according to the manufacturer’s instructions. A Custom CDF V. 22 with ENTREZ-based gene definitions was used to annotate the arrays.23 (link) The raw fluorescence intensity values were normalized by applying quantile normalization and robust multiarray analysis background correction. A batch normalization was used to remove the individual mouse variations. An analysis of variance was performed to identify differentially expressed genes using the commercial software package SAS JMP Genomics, V. 7 (SAS Institute). A false positive rate of a=0.05 with false discovery rate correction was taken as the level of significance. Gene set enrichment analysis was used to determine whether defined lists (or sets) of genes exhibit a statistically significant bias in their distribution within a ranked gene list.24 (link) Pathways belonging to different cell functions were obtained from public external databases (KEGG, http://www.genome.jp/kegg).

LncRNA and mRNA expression profiling was performed using the Affymetrix GeneChip Mouse Gene 2.0 ST array (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. Intensities of target hybridization to respective probe features were detected by laser scanning of the array. First, quantile normalization of the microarray data of the 3 untreated and 3 CRYBB2 KO mice was performed. The data was then log2-scale transformed. Hierarchical clustering of the lncRNA and mRNA profiles was performed using Cluster 3.0 software (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm) (25 (link)). The normalized expression values of the lncRNAs and mRNAs were centered on the median before unsupervised hierarchical clustering was performed. Clustering was performed with complete linkage and centered Pearson correlation. To estimate the accuracy of the measurements, the coefficient of variance for each measured parameter was determined.

Following treatment, RNA was isolated from primary chondrocytes using TRIzol™ (Invitrogen) as per manufacturer's instructions and as previously described [19 (link)]. In brief, cell homogenization with TRIzol™ was followed by the addition of chloroform for phase separation and the upper aqueous phase was removed. RNA was precipitated using 100% isopropanol and washed with 70% ethanol. RNA was allowed to air-dry prior to resuspension in RNAse-free water, then quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). RNA integrity was determined using the Agilent 2100 Bioanalyzer, and samples with RNA integrity number (RIN) greater than 8 were used for microarray analyses at the London Regional Genomics Centre, Robarts Institute (London, ON, Canada).
Total RNA (200 ng/uL per sample) from three independent cell culture experiments per treatment underwent 2 rounds of amplifications, then labeling and hybridization to Affymetrix GeneChip™ Mouse Gene 2.0 ST Array (Thermo Fisher Scientific) containing probe sets representing > 28,000 coding transcripts, as previously described [19 (link)]. The complete microarray data set will be deposited at the publicly accessible Gene Expression Omnibus (GEO) database, and NCBI accession number provided.