Genechip 3 ivt express

RNA was processed for hybridization with GeneChip 3′ IVT Express (Affymetrix - Santa Clara, USA), in accordance with the manufacturer’s instructions. Briefly, cDNA was synthesized from immunoprecipitated RNA by reverse transcription, followed by second-strand synthesis to generate double-stranded cDNA. An in vitro transcription reaction was used to generate biotinylated cRNA. The cRNA was purified and fragmented, and then hybridized onto GeneChip Affymetrix Human Genome U133 Plus 2.0 arrays. Post hybridization washes were performed with an Affymetrix GeneChip® Fluidics Station 450. Arrays were scanned on an Affymetrix GeneChip® Scanner 3000. Scanned arrays were normalized with the GCRMA in Partek software (Partek Incorporated. St. Louis, MO). Signal intensity ratios were calculated and a list of genes displaying a fold-change in abundance (IP-DZIP1/IP-negative) of at least 2.0 was generated. The list obtained was used as an input for Ingenuity pathway analysis (IPA), to determine the functional relationships between the mRNA enriched in DZIP1 immunoprecipitations. All microarray data were submitted to the GEO database and can be found under accession number GSE28882.

RNA was isolated from ~ 200 mg of VAT (N = 30) using the RNeasy Lipid Tissue Mini Kit (Qiagen). RNA quality and quantity were analyzed using a NanoDrop spectrophotometer as described above, with all samples having 260:280 ratios > 1.8. Global VAT gene expression was analyzed using Affymetrix Hu133 Plus 2.0 microarrays (Affymetrix, Santa Clara, CA; Accession: GSE88837). Briefly, extracted RNA was twice amplified using Affymetrix GeneChip 3′ IVT Express per manufacturer protocol. Biotinylated cRNA (30 μg) was hybridized to the microarrays. For resultant data, CEL files were imported into Affymetrix Expression Console and CHP files were generated using the PLIER (Probe Logarithmic Intensity Error) algorithm (Affymetrix). Standard quality control measures for adequate amplifications, thresholds for appropriate scaling factors, and RNA integrity (GAPDH 3′/5′ and HSAC07 3′/5′) were used [20 (link)]. Samples not meeting quality control standards at any point in the above-described process were reprocessed from original total RNA.

All procedures were performed at Boston University Microarray Resource Facility as described in the Affymetrix GeneChip 3′IVT Express user manual (Affymetrix, Santa Clara, CA, USA; www.affymetrix.com). For mRNA arrays biotin-labeled amplified total RNA was purified, fragmented, and hybridized to Affymetrix U133A 2.0 microarrays. The MAS5 algorithm with global scaling normalization was used to generate gene-level expression data. For miRNA arrays miRNA was purified, fragmented and hybridized to Affymetrix GenChip miRNA 3.0 microarrays. The signal of the samples was amplified and microarrays were immediately scanned using Affymetrix GeneArray Scanner 3000 7G Plus (Affymetrix, Santa Clara, CA, USA). The resulting CEL files were summarized using the Affymetrix Expression Console (current version 1.1). Clustering was performed using Cluster 3.0 software. Both mRNA and miRNA Affymetrix data [GEO:GSE81294] and SubSeries linked to GSE81294 [GEO:GSE81292; GEO:GSE81293] are available at the public repository Gene Expression Omnibus. Additional file 1: Table S1 and Additional file 2: Table S2 contain all the miRNA and mRNA, respectively, that were significantly different in patients with SSc-ILD and controls.