Ingenuity pathways analysis ipa

Pathway enrichment analysis was performed using Qiagen’s Ingenuity Pathways Analysis (IPA, Ingenuity Systems Inc.). The software determines the significance (p value) of a particular pathway being represented in the list of differentially expressed genes by Fisher’s exact. IPA is also able to show the canonical pathway participated by any of the dysregulated genes, the gene functions, and the potential gene network interactions.
A dataset with gene identifiers and corresponding FC values were entered into IPA software. These molecules were overlaid onto a global molecular network (contained in the Ingenuity Knowledge Base) during the analyses. Default settings were used to perform the analyses. The functional analysis in IPA allows determination of the biological functions and diseases associated with each network. Since the data composition of Ingenuity Knowledge Base can change over time, the results of the IPA analyses performed at different times may differ in the details uncovered.

The Ingenuity Pathways Analysis (IPA) (Ingenuity Systems, Redwood City, CA) software program was further used to determine the functional pathways represented by the identified genes. The IPA software package contains a database of biological interactions among genes and proteins, which was used to calculate the probability of a relationship between each canonical pathway, upstream pathway and the identified proteins. The IPA program scans the set of input proteins to identify networks using the Ingenuity Pathway Knowledge Base (IPKB) for interactions between identified proteins and known and hypothetical interacting genes stored in the IPA software database.

Microarray analysis was performed as described before [17 (link)]. Briefly, control and transplanted kidney tissue were used for total RNA isolation using Qiagen RNAeasy total RNA isolation kits (Qiagen, Hilden, Germany). The used tissue covered all segments of the kidneys, including cortex, medulla, and papilla. The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus [24 (link)] and are accessible through GEO series accession number GSE6497. Identification of significantly different expressed genes between the allogeneic and syngeneic groups was performed by class comparison using BRB-ArrayTools, developed by Dr. Richard Simon and Amy Peng [25 ]. The type of univariate test used was a two-sample t-test. Exact multivariate permutations tests were computer-based, performed on 126 available permutations. Nominal significance level of each univariate test was set to 0.001. Confidence level of false discovery rate assessment used was 90%, and the maximum numbers of false-positive genes allowed were set to 10. To illustrate the signalling pathways with affected genes in the AR group, the data were analysed and visualised with Ingenuity Pathways Analysis (IPA; Ingenuity Systems, Redwood City, CA, USA).

The functional annotation of differentially expressed genes was performed using Blast 2 GO as follows: i) an initial annotation with BLASTX (against the non-redundant NCBI database; e-value at 1.10⁻⁶); ii) assignment of Gene Ontology terms (GO; http://www.geneontology.org/); iii) protein domain searches using InterProscan; and iv) enzyme annotation using the Kyoto Encyclopedia of Genes and Genomes (KEGG). GO term enrichment was performed on the list of genes differentially expressed between diploid and triploids according to a Fisher exact test (p<0.05).
Data were further interpreted using Ingenuity Pathways Analysis (IPA) (Ingenuity Systems, Redwood City, CA, USA) (http://www.ingenuity.com). The list of significantly regulated genes, as selected by the statistical analysis described above, was loaded into IPA to generate networks of genes associated with particular biological functions and molecular processes.

Two tools were used for this analysis: the Functional Enrichment Tool (FET), developed by ALMAC Diagnostics based on Gene Ontology (GO) annotations [26 (link)], and the commercial software Ingenuity Pathways Analysis (IPA, Ingenuity Systems, www.ingenuity.com). The analyses enabled the identification and organization of biological entities associated with the lists of differentially expressed genes [27 (link)]. Each entity was organized according to the statistical value obtained in the enrichment analysis [28 (link)], which was adjusted for “multiple testing” [24 ]. This analysis was used to evaluate the probability that the association between a specific gene and a biological entity was random.