Genespring version 12

Total adipose tissue RNA was extracted using a total RNA Isolation Kit (mirVana™, Ambion, Sao Paulo, SP, Brazil). Six microarrays were performed from the CD–CD group (n=3) and LCD–CD group (n=3). Briefly, 300 μg of total RNA was reverse transcribed into cDNA. Samples were then transcribed into cRNA and labelled with the fluorescent dye Cy3 (for test sample) or Cy 5 (for reference sample). Then, the samples were hybridized to an Affymetrix GeneChip Mouse Gene 2.0 ST whole transcript-based array (Affymetrix, Santa Clara, CA, U.S.A.). This gene array includes 26515 genes. The slides were then scanned by Affymetrix GeneChip Command Console software (Affymetrix, Santa Clara, CA, U.S.A.). All data were transferred into GeneSpring version 12.5 (Agilent Technologies, Palo Alto, CA, U.S.A.) to normalize the data, control the quality and analyse. Fold change and P value (from t test) were used to identify differentially expressed genes (DEGs). The threshold was set at a fold change ≥1.5 and a P value ≤0.05.

Data files generated by the Agilent G2565CA Microarray Scanner System (Agilent Technologies) were imported into the GeneSpring version 12.5 (Agilent Technologies). Normalized data from all samples were filtered on genes flagged as present or marginal with the resulting gene list used for further gene expression analysis and clustering (Student's t-test against zero, asymptotic P-value computation and Benjamini Hochberg FDR multiple testing correction). Changes were expressed as the ratio of gene expression of wt FA1090 strain over expression of the same gene for the mutant. Genes with differential expression of ≥1.5-fold and a P-value < 0.05 were selected. Supporting microarray data for all studied gonococcal mutant strains have been deposited in the NCBI's Gene Expression Omnibus (Edgar et al., 2002 (link)), and are accessible through GEO series accession number GSE60347 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE60347).