Agilent feature extraction software 9

The hybridization images were analyzed by an Agilent DNA Microarray Scanner (Agilent Technology) and data quantification was performed using Agilent Feature Extraction software 9.3.2.1 (Agilent Technology) as described previously [14 (link)]. The average fluorescence intensity for each spot was calculated and the local background was subtracted. All data normalization and the selection of fold-changed genes were performed using GeneSpringGX 7.3.1 (Agilent Technology). The genes were filtered to remove flag-out genes in each experiment. Intensity-dependent normalization (LOWESS) was performed, where the ratio was reduced to the residual of the Lowess fit of the intensity vs. ratio curve. The average of the normalized ratios was calculated by dividing the average of the normalized signal channel intensity by the average of the normalized control channel intensity. Genes changed > 2.0-fold were selected and considered significant genes. The microarray result has been deposited into the Gene Expression Omnibus (GEO; GSE158940).
The functional annotation of genes was performed according to Gene OntologyTM Consortium (http://www.geneontology.org/index.shtml) by GeneSpringGX 7.3.1.

To determine candidate genes, whose expression is altered upon TSN silencing, RNA was extracted from both mock-transfected and TSN siRNA-transfected A549 cells using a PureLink™ RNA Mini Kit (Life Technologies (Thermo Fisher Scientific), Carlsbad, CA, USA). Four biological replicates were considered to perform a gene expression analysis. Samples were hybridized to Agilent G4851A SurePrint G3 Human Gene Expression 8 × 60 K Microarray slides (listed with 27, 958 target gene RNAs and 7, 419 lincRNAs: Design ID 028004, Agilent Technologies, Santa Clara, CA, USA). All arrays were scanned by Agilent Microarray Scanner (G2565BA, Agilent Technologies, Santa Clara, CA, USA) and subsequently analyzed by Agilent Feature Extraction software 9.5.3.1 (Agilent Technologies) and Gene Spring GX12.0.2 software (Agilent Technologies, Santa Clara, CA, USA). Probes with an average of 2.0-fold changes were designated as TSN-regulated genes. The Ingenuity Pathways Analysis (IPA) program (Ingenuity Systems, Mountain View, CA, USA; http://www.ingenuity.com) was used to identify networks and canonical pathways of genes that appeared differentially expressed after silencing of TSN. With use of the Ingenuity knowledge database, the genes closely associated with cell death and survival pathways were selected for investigation.

Genomic DNA was quantified by spectrophotometry (NanoDrop ND1000, NanoDrop Technologies, Wilmington, Delaware USA). Integrity of DNA was assessed by 0.8% agarose gel electrophoresis. Non-amplification labeling of DNA (direct method) was obtained following the ‘Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis’ protocol Version 4.0 (Agilent Technologies, Palo Alto, California USA. p/n G4410-90010). 500 ng of experimental and pool female reference genomic DNA samples were fragmented in a restriction digestion step. Digestion was confirmed and evaluated by DNA 7500 Bioanalyzer assay. Cyanine 3-dUTP and cyanine 5-dUTP were used for fluorescent labeling of test and reference digested gDNAs respectively, using the ‘Agilent Genomic DNA Labeling Kit PLUS’ (Agilent p/n 5188-5309) according to the manufacturer's instructions. Labeled DNA was hybridized with Human Genome CGH Microarray 44K (Agilent p/n G4426B-014950) containing 43,000+ coding and noncoding human sequences. Arrays were scanned in an Agilent Microarray Scanner (Agilent G2565BA) according to the manufacturer's protocol and data extracted using Agilent Feature Extraction Software 9.5.3.1 following the Agilent protocol CGH-v4_95_Feb07 (‘Lowess Only’ normalization correction dye bias method instead of ‘Linear Only’) and the QC Metric Set CGH_QCMT_Feb08.

Samples were shipped to Agilent Array Services (Hokkaido System Science, Sapporo, Japan). RNA was amplified into cDNA and labeled according to the Agilent One-Color Microarray-Based Gene Expression Analysis Protocol (Agilent Technologies). The samples (n = 3) were hybridized to Rat GE 4x44K v3 array slides, and the arrays were then scanned using an Agilent Microarray Scanner (Agilent Technologies). The scanned images were analyzed using the standard procedures described in the Agilent Feature Extraction software 9.5.3.1 (Agilent Technologies).
To compare rat and mouse genes, rat orthologs were checked manually in PubMed (http://www.ncbi.nlm.nih.gov/pubmed) or the Rat Genome Database (http://rgd.mcw.edu/). We excluded a gene from the lists if no rat ortholog was found, or if it was not listed in the Agilent Rat GE 4x44K v3 array. When more than one expression value was obtained for a gene, the largest expression value was used. Complete lists of the cell-type-specific genes are provided in Supplementary Data 1 and 2; complete lists of the top genes are listed in Fig. 1f and Supplementary Fig. 2.