Genespring software version 12

After the completion of microarray analysis, data were subjected to summarization, normalization and quality control by using GeneSpring software version 12.0 (Agilent, CA, USA). Differentially expressed genes were defined as those with a fold change equal or greater than two and a Benjamini–Hochberg corrected P value less than 0.05. The expression level of each gene was log 2 transformed and median centered by employing the Multiexperiment Viewer software (Dana-Farber Cancer Institute, MA, USA).

To compare the gene expression profiles in photoreceptor-directed fibroblasts derived from EYS-RP patients with those from normal volunteers, we analyzed the expression levels of 58,201 probes in the induced/non-induced photoreceptor-like cells with or without EYS genes defects using the SurePrint G3 Human Gene Expression Microarray 8 × 60 K Ver.2.0 (Agilent Technologies, Palo Alto) using total RNA extracted from the cells. To average experimental variations, extracted total RNA samples were pooled into one tube from three independent induction experiments, and pooled samples were subjected to microarray analyses. To normalize the variations in staining intensity among chips, the 75th percentile of intensity distribution was aligned across arrays using GeneSpring software version 12.5 (Agilent). We first compared expression profiles for all the probes and then for refined probes based on GO terms: retina-related and apoptosis, oxidative stress, ER stress or aging (GO (retina) and GO (cell death), respectively). We extracted the intersection of two groups of genes, i.e., up- or downregulated and GO (retina) or GO (cell death)-related genes. For pathway enrichment analysis, WebGestalt2017 (http://www.webgestalt.org/) with KEGG pathway (Kyoto Encyclopedia of Genes and Genomes pathway) as a database was used.

To interrogate the molecular mechanism underlying P4HB's actions, we studied the gene expression profiles of 73 human specimens from Genomic Spatial Event (GSE) 16011 at the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/). These included 7 normal controls, 17 World Health Organization (WHO) grade II, 29 grade III, and 20 grade IV gliomas. The raw CEL files generated from Affymetrix GeneChip Human Genome U133 Plus 2.0 Array were Robust Multi-Array Average (RMA) preprocessed and normalized using GeneSpring software version 12.5 (Agilent Technologies, Santa Clara, CA, USA). The differential gene expression profiles of tissues with low- or high-P4HB expression (using the median expression level as cut-off) were identified. GO enrichment analysis was performed to delineate the predominant functions of up-regulated genes (p ≤ 0.00001) under the GO category ‘biological processes’.

Rice seedlings were collected after 2 days of ‘Cr’ and ‘CrP’ treatments to wash with deionized water and separate their shoots and roots. Subsequently, total RNA was isolated from both the roots and shoots of treated rice seedlings as described in Yu et al. [17 (link)]. Briefly, Ultrapure RNA Kit [(DNase I) (CWBio, Taizhou, China)] was used to extract total RNA from the 0.2 g of rice tissues. To prevent any genomic DNA contamination, the extracted RNA was initially treated with DNase I and then purified using RNeasy MinElute Cleanup kit (Qiagen, Helden, Germany), respectively.
The Agilent 4X44K rice microarray with oligonucleotide 44,000 probes was used in this study. Microarray hybridization, washing, staining, scanning, and data processing were carried out by Shanghai Biotechnology Corporation (Shanghai, China). Hybridization signal data were deposited into GeneSpring Software version 12.5 (Agilent Technology, Santa Clara, CA, USA) for screening of differentially expressed genes (DEGs). The DEGs data were normalized by Quantile algorithm. The maximum level for the selection of DEGs was fixed as a p value < 0.05 and the fold change ratio between the non-treated and treated samples was <0.5 or >2.0 [17 (link)].