Command console

Human full-genome Affymetrix GeneChip HG-U133 Plus 2.0 (PartnerChip, Evry, France) were used following the manufacturer’s recommendations. These microarrays contain 55000 probe sets (25 nucleotides per set) covering 30000 transcripts. Briefly, RNA quality and quantity were estimated using the Nanodrop (ND-1000) and BioAnalyzer 2100 systems (Agilent, Les Ulis, France). When too-high concentrations of salts or solvents were detected, RNA precipitation and washing were performed before sample processing. Quantification of array fluorescence signals was carried out using a GeneChip 3000 scanner. Then, array data were analyzed using the Affymetrix Command Console software. Quality control and statistical analyses were performed using the Affymetrix Expression Console and GeneSpring GX11 softwares.

Total RNA was extracted from 20–30 mg powdered (in liquid nitrogen) whole heart tissue using the Rneasy Fibrous Kit (Qiagen), including a DNase treatment step. The RNA integrity was assessed on a BioAnalyzer (Agilent Laboratories); all samples had a RNA Integrity Number ≥7. Labeled sense single-strand DNA (ssDNA) for hybridization was generated from 200 ng starting RNA with the Ambion WT expression kit (P/N 4411973) and the Affymetrix GeneChip WT Terminal Labeling and Controls Kit (P/N 901525) according to the manufacturer's instructions. The distribution of fragmented sense ssDNA lengths was measured on the BioAnalyzer. The fragmented ssDNA was labeled and hybridized for 17 h at 45°C to the Affymetrix GeneChip Human Mouse 1.0 ST Array (Affymetrix). Chips were processed on an Affymetrix GeneChip Fluidics Station 450 and Scanner 3000. Affymetrix Command Console was used to generate cel files, and Affymetrix Expression Console was used for the quality control. Arrays were RMA normalized in GeneSpring GX 12, and differentially expressed genes were identified using Limma with a Benjamini and Hochberg multiple testing correction of ≤0.05. Because few genes were detected using this method, the data were reanalyzed using PLIER normalization and a Student's t-test with a P value cutoff of ≤0.05 and a fold change difference between WT and Chuvash of ≥1.3.

Prior to study commencement, the Health Research Ethics Committee for the North Denmark Region approved our study protocol (MSCNET, N-20080062MCH). Following informed written consent, obtained in accordance with the Declaration of Helsinki, normal BM was collected from the sternum of seven adult patients undergoing cardiac surgery as described in the S1 Appendix, and elsewhere [12 (link)]. Fluorescence-activated cell sorting (FACS) was used to fractionate mononuclear BM cells into six distinct normal B-cell subsets: pre-BI (BI), pre-BII (BII), immature (IM), naïve (NA), memory (ME), and plasma cells (PC) (S1 Appendix and S1 Table). For gene expression profiling (GEP), mRNA from B-cell subsets were isolated and hybridized to the Human Exon 1.0 ST (Exon) array platform [12 (link)]^. A total of 38 CEL files containing B-cell data were generated using the Affymetrix Command Console. The CEL files and metadata were adjusted for compliance (with the requirements of Minimum Information About a Microarray Experiment) [16 (link)], and then deposited in the NCBI Gene Expression Omnibus repository (accession code GSE68878). In the present study, these data are referred to as the sternal BM B-cell data.

The Mouse Gene 2.1. ST Array (Affymetrix, Schwerte, Germany) was hybridized with RNA isolated from normal and tumorous liver tissues of control- and chemerin-156-infected mice (five animals per group). The Ambion WT Expression Kit and Affymetrix WT Terminal Labeling and Hybridization procedure were used according to the suppliers´ suggestions. Data were analyzed using the Affymetrix Command Console and Expression Console. Differences were calculated by the unpaired Student´s t-test (Kompetenzzentrum für Fluoreszente Bioanalytik, Regensburg, Germany). After Bonferroni correction, not a single gene was significantly changed in the tumor when compared to the respective non-tumorous tissues of control-AAV-infected animals. Real-time RT-PCR analysis revealed that Spink1 was significantly induced in the tumors and the respective p-value for this difference (p = 0.01289) was chosen as cut off value. Principle component analysis and cluster dendrogram were performed as described [79 ,80 (link)].