Feature extraction software

Fluorescence signals from hybridized microarrays were detected using an Agilent and DNA microarray scanner with a resolution of 5l M and using Agilent Feature Extraction Software (FES). FES determines feature intensities and normalized ratios by linear LOWESS with background subtraction, rejects outliers and calculates statistical confidences (P-values). Hybridization signals with P value less than 0.001 were considered significant. Only genes differentially expressed in the three repeat experiments were considered as relevant genes.

This protocol is fully described in Cayre et al., 2013 (link). Briefly, OPC from eight mice induced for experimental autoimmune encephalomyelitis (EAE mice) at the peak of paralytic symptoms and from eight adult healthy mice as controls were purified using magnetic cell sorting (Miltenyi Biotec). This experiment was replicated in an independent similar experiment. cDNAs were prepared and used (250 ng) as template for Cy3 and Cy5, combined and hybridized to Agilent Whole Mouse Genome Oligo Microarrays 4 Å~44K. Agilent Feature Extraction Software (FES) determines feature intensities and ratios (including background subtraction and normalization), rejects outliers and calculates statistical confidences (P-values). We obtained a gene list with all normalized Cy5/Cy3 log10 ratios, Cy5/Cy3 fold changes, sequence description and P-values. Microarray data are available at GEO with accession number GSE47486.

250 ng of each of the cDNAs were used as template for Cy3 labeling which was performed according to Miltenyi Biotec’s undisclosed protocol. The Cy3- labeled cDNAs were hybridized overnight (17 hours, 65°C) to an Agilent Whole Human Genome Oligo Microarrays 4 x 44K using Agilent’s recommended hybridization chamber and oven. Finally, the microarrays were washed once with 6x SSPE buffer containing 0.005% N-lauroylsarcosine for 1 min at room temperature followed by a second wash with pre-heated 0.06x SSPE buffer (37°C) containing 0.005% N-lauroylsarcosine for 1 min. Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (Agilent Technologies). The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files. For determination of differential gene expression FES derived output data files were further analyzed using the Rosetta Resolverâ gene expression data analysis system (Rosetta Biosoftware). Ratios were calculated by dividing sample signal intensity through control signal intensity (automated data output of the Resolverâ system).

Plasmatocytes were isolated from w;HmlΔ-Gal4,UAS-2xeGFP flies that were either kept for 30 days on lard-supplemented food or normal food as described above. Five thousand to ten thousand GFP-positive plasmatocytes were FACS sorted from five independent samples per group and sorted directly into 6.4 μl preheated SuperAmp Lysis buffer (Miltenyi). Samples were incubated for 10 min at 42°C according to the manufacturer’s guidelines and stored at −20°C until shipping. Samples were further analyzed and processed by the Miltenyi Genomic Service. For each of the cDNAs, 250 ng was used as template for Cy3 labeling according to the manufacturer’s protocol. The Cy3-labeled cDNAs were finally hybridized to an Agilent Whole Drosophila Genome Oligo Microarrays Custom 8x60K. The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files. The software determines feature intensities (including background subtraction), rejects outliers, and calculates statistical confidences. Normalized data sets were compared using an unpaired t test (p < 0.01) and a fold change > 2 between normal diet and lard-supplemented food. Annotated significantly differentially regulated genes were depicted in a heat map. Heat map of differentially regulated genes represents the log₂ of the fold change between normal diet and lard-supplemented diet.