Ingenuity pathway analysis software

Cellular function and localization of identified genes and proteins were obtained with the Ingenuity Pathway Analysis software (Ingenuity Systems, US).
Lists of genes or proteins significantly induced or repressed after exposure to NPs were uploaded into Ingenuity Pathway Analysis software for biological analysis by comparison with the Ingenuity Knowledge Database. These lists of altered genes or proteins were then processed to investigate their functional distribution, as defined by Gene Ontology. Datasets were analyzed with several tools such as SecretomeP, SignalP, Exocarta and Vesiclepedia. Known canonical pathway associations were measured by IPA by using a ratio of the number of genes or proteins from datasets that map to the pathway divided by the total number of constitutive genes or proteins that map to the canonical pathway. A Fisher’s exact test was used to determine a p-value representing the significance of these associations.

The Top 1000 DEGs (based on the p values) obtained from comparing whole blood at cold vs the frozen conditions were uploaded into Ingenuity Pathway Analysis software (Ingenuity Systems, Redwood City, CA) and mapped to the functional networks available in the Ingenuity Pathway Knowledge Base. The expression analysis using IPA was done using default settings and stringent filter for tissues and primary cells settings. The top physiological system and biofunctions of the core analysis was conducted using the default settings for our dataset. P-values were corrected for multiple testing using the Benjamini-Hochberg (B-H) false discovery rate determining the association between the genes in the dataset and the biofunctions these are associated.

The total RNA from cells was isolated using peqGOLD TriFast™ according to the manufacturer’s protocol. RNA samples were purified using the RNA Clean-Up and Concentration Micro Kit, RNA concentration was determined using spectrophotometry (NanoDrop 8000), and quality control was performed using an Agilent 2100 Bioanalyzer. Microarray analysis was carried out using individual RNA samples (n = 4), which were processed following the manufacturer’s instructions of the GeneChip^TM WT PLUS Reagent Kit (Thermo Fisher Scientific) and hybridized with GeneChip^TM Clariom D Rat Arrays (Thermo Fisher Scientific). Quality control of the hybridizations and data analysis were performed using Transcriptome Analysis Console (Thermo Fisher Scientific). The data were normalized using the Robust Multi-chip Analysis (RMA) algorithm.
To identify significantly differentially expressed genes (q–value < 0.05, fold change ≥ 1.3-fold), one-way ANOVA (with Bayes estimation) was performed, where p-values were corrected for multiple testing using Benjamini Hochberg adjustment. All data from transcriptome analyses are provided in Table S1. Significantly differentially expressed genes were further subjected to in silico pathway analysis using the Ingenuity Pathway Analysis software (Ingenuity Systems, Inc. Redwood City, CA, USA). This enrichment analysis was based on all annotated rat genes in the database.