Affymetrix transcriptome analysis console

We downloaded raw gene expression data from the US National Center for Biotechnology Information (NCBI) GEO available online at: https://www.ncbi.nlm.nih.gov/geo/. The samples (filename GSE53072_RAW.tar) were categorized into two groups: the control group comprising data from three normal thyroid glands (GSM1281638, GSM1281639, and GSM1281640) and the thyroid cancer group comprising data from five thyroid carcinoma groups (GSM1281636, GSM1281637, GSM1281641, GSM1281642, and GSM1281643). We performed Affymetrix Human Genome U133 Plus Version 2.0 Array (GPL570) analysis using the Affymetrix Transcriptome Analysis Console (both from Affymetrix, Santa Clara, CA, USA). Differentially expressed miRNAs/mRNAs were considered those that met the following criteria: P < 0.05 and log₂|fold change (FC)|> 1.5. We drew a heatmap and a volcano plot using the results of differentially expressed miRNA/mRNA analysis. We used data from TCGA-THCA to analyze the disregulated mRNAs and the TargetScan database (version 7.2; http://www.targetscan.org/vert_72/) to investigate potential downstream miRNA targets.

RNA was isolated from sperm collected from the cauda epididymis and purified using an RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA). Total RNA (100 ng) was used to synthesise double-stranded cDNA (dsDNA). Anti-sense cRNA was synthesized from the dsDNA template and subsequently used to produce sense single-stranded cDNA (ssDNA). The ssDNAs were fragmented, end-labelled, and hybridised to a Genechip Mouse Transcript Assay 1.0 set (Affymetrix, Tokyo, Japan). Arrays were hybridised and scanned using a standard protocol. Microarray data were analysed using an Affymetrix Expression Console and Affymetrix Transcriptome Analysis Console (Affymetrix, Tokyo, Japan).

We downloaded raw gene expression data from the US National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/). The samples (filename GSE40885_RAW.tar) were divided into two groups: seven alveolar macrophages from lung subsegments treated with LPS (GSM1004102, GSM1004104, GSM1004106, GSM1004108, GSM1004110, GSM1004112 and GSM1004114) and seven alveolar macrophages from lung subsegments instilled with saline solution (GSM1004101, GSM1004103, GSM1004105, GSM1004107, GSM1004109, GSM1004111 and GSM1004113). We analysed the Affymetrix Human Genome U133 Plus Version 2.0 Array (GPL570) using the Affymetrix Transcriptome Analysis Console (both from Affymetrix). Differentially expressed lncRNAs/mRNAs were identified as having P < .05 and |fold change (FC)| > 2. We drew a heatmap and a volcano plot using the results of differentially expressed mRNA analysis.

For in vitro analyses, one-way ANOVA was performed. The log rank test was used to compare patient survival in the primary breast cancer cohort. Analyses were performed using GraphPad Prism version 7.03 (GraphPad Software, La Jolla California, USA). For Affymetrix analyses, ANOVA P-values were calculated using the Affymetrix Transcriptome Analysis Console (Affymetrix). The time course plots were plotted as fold-change with the baseline value set at 1.

Total RNA from NF-κB-silenced and non-silenced cells was obtained using the RNeasy Mini kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Next, 100 ng of each RNA was used to synthesize and biotinylate cRNA according to the GeneChip whole transcription (WT) sense target-labeling assay (Thermo Fisher). The biotinylated cRNA was hybridized to the GeneChip human gene 1.0 ST array (Thermo Fisher), washed and stained according to the manufacturer’s protocols. The GeneChip arrays were scanned using GeneChip^® Scanner 3000. Affymetrix Expression Console™ software v.1.0 (Thermo Fisher) was used to create summarized expression values (CHP-files). Data were analyzed using the Affymetrix Transcriptome Analysis Console (Thermo Fisher), whereby differentially expressed genes with ≥1.5-fold-change were used as criteria to define up-regulation or down-regulation compared with the corresponding Scramble.
In silico analysis of gene ontology, biological processes and signaling pathways, that were altered as a consequence of NF-κB knockdown, was performed using KEGG [19 ] and PANTHER software [20 (link)]. A pathway enrichment analysis was performed using MetaCore software v.6.35 (Thomson Reuters, Toronto, Canada) A Venn diagram was generated using the online software of Bioinformatics & Evolutionary Genomics [21 ].