Computational protocol: Concurrent progress of reprogramming and gene correction to overcome therapeutic limitation of mutant ALK2-iPSC

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Protocol publication

[…] Total RNA from human iPSCs was extracted using TRIzol and the RNAeasy Mini Kit, then amplified and labeled using the Low RNA Input Linear Amplification kit PLUS (Agilent Technologies) and then hybridized to the human whole genome 44K array (Agilent Technologies). The arrays were scanned using an Agilent DNA Microarray Scanner and the raw intensity of the probe signals was extracted using Feature Extraction Software (Agilent Technologies). Only probes showing signal intensities greater than 1.4 times the local background were selected; these were normalized using the quantile method. Gene Ontology (GO) network analysis was performed using the REVIGO program. Initially, using differentially expressed genes with at least twofold variation, simple enriched GO terms were obtained from the Functional Annotation Tool of DAVID in which the P-value of each GO term is calculated using Fisher's exact test and is adjusted for multiple comparisons using the Benjamini–Hochberg procedure. Thereafter, GO terms with false discovery rates <0.001 were selected as simple enriched GO terms that were used as input data for the two network programs. In the GO term network structure obtained from REVIGO, the node size and color intensity are proportional to the hierarchical status and statistical significance of each node, respectively. The edge thickness between nodes represents the closeness of the two nodes. Data are publicly available in the GEO database (http://www.ncbi.nlm.nih.gov/geo, accession number GSE68766). For analysis of other public microarray data, CEL files from GSE61510 were imported into Affy R package and normalized using Robust Multi-array average (RMA) algorithm. […]

Pipeline specifications

Software tools REViGO, affy
Application Gene expression microarray analysis
Organisms Homo sapiens
Diseases Myositis Ossificans