Hg u133 plus 2.0 genechip microarrays

Human atherosclerotic carotid artery lesions were obtained from patients undergoing endarterectomy surgery for stable (asymptomatic) (n = 40) or unstable (symptomatic) (n = 87) carotid stenosis, as part of the Biobank of Karolinska Endarterectomies (BiKE). Normal control arterial samples (n = 10) were obtained from the iliac and radial arteries from healthy organ donors without any history of cardiovascular disease. Briefly, tissue was snap frozen in liquid nitrogen before pulverizing to a fine powder using a pre-chilled mortar and pestle, then resuspended in Qiazol lysis reagent (Qiagen) and homogenized with a rotor stator tissue homogenizer. Total RNA was extracted as described above using the miRNeasy Mini Kit (Qiagen) and RNA quality assessed using a Bioanalyzer 2100 (Agilent). Global gene expression profiles were analyzed by Affymetrix HG-U133 plus 2.0 Genechip microarrays from 127 patient derived plaque samples and 10 donor control samples. Robust multi-array average (RMA) normalization was performed and processed gene expression data presented in Log2 scale. For TaqMan based analysis, miRNA-specific cDNA was prepared as described above, and TaqMan qPCR was performed in triplicates using predesigned TaqMan probes for miR-224 and normalized to the RNU44 internal control. Data are represented as mean Log2 fold change of replicates from two independent experiments.

RNA from sorted MM and HD BM-MSC was extracted using the RNeasy Minikit (Ref: 74106, Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Then, cDNA synthesis, in vitro transcription and fragmentation of cRNA were performed using the GeneChip 3′IVT PLUS Reagent kit, (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. After assessment of RNA integrity (Agilent Small RNA Analysis kits, Agilent 2100 Bioanalyser, Agilent, Santa Clara, CA, USA), biotinylated RNA was hybridized with the Affymetrix HG-U133 plus 2.0 GeneChip microarrays (Affymetrix, Santa Clara, CA, USA), and analysis was performed as described previously. Raw Affymetrix cell intensity files (.CEL) were used for the differential expression analysis.

RNA from the NPC cells treated with 200 nM PDPN siRNA was amplified and labeled with biotin using the Ovation Biotin RNA Amplification and Labeling System (NuGEN, San Carlos, CA, USA) according to the manufacturer's procedure. The fragmented, biotinylated cDNA was used for hybridization at 45°C for 17 hours with Affymetrix HG-U133 Plus 2.0 GeneChip microarrays (Affymetrix, Santa Clara, CA) as described by the manufacturer's recommendations. We scanned and digitized the hybridization signals using an Affymetrix 7G Gene Chip Scanner and GCOS Version 1.4.0.036 software, respectively. The raw expression data for differential expression analysis were normalized using the GCRMA package and the expression value for each probe was defined as the base 2 logarithm of the intensity in the samples. The microarray data were deposited in the GEO database (GEO accession number: GSE128502). We then used Ingenuity Pathway Analysis (IPA) to assign biological functions to genes and network analysis using the Ingenuity Pathways Knowledge Base (Ingenuity Systems, Inc., Redwood City, CA, USA).

Late stage/Advanced atherosclerotic plaques were obtained from patients undergoing surgery for high grade (>50%) carotid stenosis and retained within the BiKE study. Normal artery controls were obtained from nine macroscopically disease-free iliac arteries and one aorta from organ donors without history of cardiovascular disease. All samples were collected with informed consent from patients or organ donor guardians. 127 plaques from BiKE patients and 10 normal arteries were analyzed by Affymetrix HGU133 plus 2.0 GeneChip microarrays. Robust multiarray average normalization was performed and processed gene expression data was transformed in log2-scale. The microarray dataset is available from Gene Expression Omnibus (GSE21545). The BiKE study cohort demographics, details of sample collection, processing, and analyses were previously described[16 (link)].

Total RNA was extracted using TRIzol reagent (Invitrogen) and purified by RNeasy Mini kit (QIAGEN). The yield and quality of RNA were assessed using spectrophotometry and the Agilent 2100 Bioanalyzer (Agilent Technologies). Microarray experiments including cDNA preparation, hybridization, scanning, and image analysis of Affymetrix GeneChip HG-U133 plus 2.0 microarrays were performed in the Microarray Core facility at Dana-Farber Cancer institute according to the manufacturer’s protocol (Affymetrix). Experiments were performed either in duplicate or triplicate.

Gene expression analysis of whole blood-derived mRNA with Affymetrix GeneChip
HG-U133 Plus 2.0 microarrays was validated previously by real-time
PCR^{[20 (link)]}.