Clariom d

Following the manufacturer’s instructions, high-quality RNA was extracted from 20 separate samples, tagged, and hybridized on human Clariom D gene chips (catalog no. 902922; Affymetrix, Santa Clara, CA, USA). The protocol was as follows: whole RNA was used to make cDNA and cRNA, which were then hybridized for 16 h at 45 °C in an Affymetrix GeneChip 645 hybridization oven, and the results were read off a human transcriptome array 2.0. A GeneChip Fluidics Station 450 was used to dye the arrays. After that, a GeneChip™ scanner 3000 was used to read the data from the chip. DAT files containing the array’s fluorescence signals were collected. Raw data in ARR and DAT image files provided the information about the pixel intensity levels. The Affymetrix GeneChip Command Console software version 4.0.1.36 was used in the process of transforming the raw data contained within the ARR and DAT image files into the intensity data that were included inside the CEL and CHP files. Affymetrix Clariom D.CEL data were normalized using Expression Console software version 1.4.1 to provide probe-level signal expression values, which were then stored as CHP files. The Transcriptome Analysis Console (version 4.0.2) was used to examine the CHP files for the expression of genes, exons, splice variants, and associated pathways involved in TAO formation [25 (link)].

One hundred ng of RNA per sample were analyzed using the ClariomD (Affymetrix) microarray assay. Library preparation, hybridization and data acquisition were performed by GENOM‘IC platform according to manufacturer‘s instructions. Gene and exon level expressions were processed and extracted from the ClariomD microarray using the Transcriptomic Analysis Console (TAC) provided by Affymetrix.

The microarray used for these studies was Affymetrix Clariom D which allows us to analyze the whole coding transcriptome at the gene and exon level as well as non-coding RNA such as lincRNA, miRNA and circRNA. Sample amplification and preparation for microarray hybridization was performed according to Affymetrix specifications according to the GeneChip WT Pico Reagent Kit protocol. Briefly, 100 ng of total RNA was reverse transcribed into cDNA, amplified by in vitro transcription and reversely transcribed to cDNA again. Fragments between 40 and 70 bp were generated enzymatically, labelled and hybridized onto the microarray chips in an Affymetrix hybridization oven at 60 rpms and 45 °C for 17 h. Chips were washed according to the stablished protocols (Affymetrix, Santa Clara, CA, USA) with a GeneChip fluidics station 450, and finally scanned with an Affymetrix 7G GeneChip scanner. The raw data (CEL files) were uploaded into the Gene Expression Omnibus (GEO), which is hosted by the National Center for Biotechnology Information (NCBI) under the accession number GSE147786.

The microarray used for these studies was Affymetrix Clariom D which allows us to analyze whole coding transcriptome at the gene and exon level as well as non-coding RNA such as lincRNA, miRNA and circRNA. Sample amplification and preparation for microarray hybridization was performed according to Affymetrix specifications. Briefly, 100 ng of total RNA was reversely transcribed into cDNA, amplified by in vitro transcription and reversely transcribed to cDNA again. Fragments between 40 and 70 bp were generated enzymatically, labelled and hybridized onto the microarray chips in an Affymetrix hybridization oven at 60 rpms and 45 °C for 17 h. Chips were washed according to the stablished protocols (Affymetrix, Santa Clara, CA, USA) with a GeneChip fluidics station 450, and finally scanned with an Affymetrix 7G GeneChip scanner. The raw data (CEL files) has been uploaded into the Gene Expression Omnibus (GEO), which is hosted by the National Center for Biotechnology Information (NCBI) under the accession number GSE147786.

Clariom™ D microarray analysis (Thermo Fisher Scientific, Waltham, MA, USA) was performed on prostate stromal cells with or without TGFβ1 treatment. To select the differentially expressed genes, we used threshold values of |Log₂FC| ≥ 1 and a Benjamini-Hochberg corrected P value of 0.05. The data were processed and analyzed following the methods described previously [51 (link)]. Finally, these differentially-expressed genes were applied for Protein-Protein Interaction Networks (STRING) analysis, KEGG pathway annotation (https://www.genome.jp/kegg/), and Gene Ontology enrichment analysis (GO analysis), and the visualization was achieved by using Cytoscape [52 (link)].