For whole transcriptome analyses, the Human Transcriptome 2.0 Array (Thermo Fisher Scientific) was used. For each microarray, the total RNA input amount was 10 ng. Sample processing has been performed following the instructions of the manufacturer. Quality control of the results was performed using Transcriptome Analysis Console software version 4.0 (Applied Biosystems, Waltham, MA).
Transcriptome analysis console software version 4
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Transcriptome Analysis Console (TAC) software version 4.0.2 is a bioinformatics application designed for the analysis of transcriptome data. It provides a suite of tools for processing and interpreting gene expression data obtained from various high-throughput sequencing platforms.
Automatically generated - may contain errors
Lab products found in correlation
Expression console software, by Thermo Fisher Scientific (2 mentions)
Genechip mirna 4.0 array, by Thermo Fisher Scientific (2 mentions)
Genechip scanner 3000 7g, by Thermo Fisher Scientific (2 mentions)
Fluidics station 450, by Thermo Fisher Scientific (2 mentions)
Human transcriptome 2.0 array, by Thermo Fisher Scientific (1 mentions)
Affymetrix gene chip hybridization oven 645, by Thermo Fisher Scientific (1 mentions)
Affymetrix genechip scanner 3000 7g, by Thermo Fisher Scientific (1 mentions)
Affymetrix genechip wt plus reagent kit, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Ingenuity pathway analysis ipa, by Qiagen (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Expression console, by Thermo Fisher Scientific (1 mentions)
Rna 6000 nano kit, by Agilent Technologies (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Genechip human exon 1.0 st array, by Thermo Fisher Scientific (1 mentions)
8 protocols using transcriptome analysis console software version 4
1
Whole Transcriptome Analysis Using Human Transcriptome 2.0 Array
2
Transcriptomic Analysis of MKN74 Cells
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MKN74 cells were seeded in 6-well plates at a density of 6.0 × 10 4 cells/well and incubated for 24 h, and the medium was replaced every 24 h with the CM (MKN74) or sEVRM (MKN74). After 72 h, the total RNA was extracted using the miRNeasy Mini Kit (Qiagen, Hamburg, Germany). Purified sense-strand cDNA was prepared from 0.1 μg of total RNA using the Affymetrix GeneChip WT PLUS Reagent Kit (Thermo Fisher Scientific). Two micrograms of cDNA were fragmented and biotinylated. Biotin-labeled cDNA was hybridized on Human Clariom S Arrays at 45 °C for 16 h at 60 rpm in an Affymetrix GeneChip Hybridization Oven 645 (Thermo Fisher Scientific). The chips were washed and scanned with an Affymetrix GeneChip Scanner 3000 7G (Thermo Fisher Scientific). Raw microarray data were processed for background corrections and quantile normalization using Affymetrix Expression Console software 1.4.1. (Thermo Fisher Scientific) and analyzed using Transcriptome Analysis Console software version 4.0 (Applied Biosystems, Foster City, CA, USA). The ingenuity pathway analysis (IPA; Ingenuity Systems, Redwood, CA, USA) was performed to analyze the signal transduction networks.
3
Profiling miRNA Expression in PeC Patients
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Samples from 11 PeC patients were used for miRNAs expression profiling using the GeneChip miRNA 4.0 Array (Thermo Fisher Scientific) following the manufacturer’s protocol. Each sample was hybridized on the array, washed, stained with the Affymetrix Fluidics Station 450, and scanned using Affymetrix GeneChip Scanner 3000 7G. Expression Console software (Thermo Fisher Scientific) was used for quality assessment. Transcriptome Analysis Console (TAC) software version 4.0.2 (Thermo Fisher Scientific) was used for background correction, normalization, and summarization of raw data (CEL files) by Robust Multichip Average plus Detection Above Background (RMA + DABG), considering probesets for mature Homo sapiens miRNAs. We used the Linear Models for Microarrays (LIMMA) test with at least two-fold changes (FC > 2), p < 0.01, and false discovery rate (FDR) < 0.05 to identify DEmiRs between TT (n = 11) and NNT (n = 11) groups. Hierarchical clustering graphs with the DEmiRs selected for further validation were also constructed using the TAC software. Microarray datasets are available on the Gene Expression Omnibus (GEO) database (accession number GSE172095).
4
miRNA Expression Profiling in Metastatic and Localized Cancers
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The miRNA expression profile was investigated in samples from patients with metastatic (n = 6) or localized disease (n = 5) using the GeneChip miRNA 4.0 Array (Thermo Fisher Scientific) following the manufacturer’s protocol. The samples were hybridized on the array, stained in the Affymetrix Fluidics Station 450, and scanned with the Affymetrix GeneChip Scanner 3000 7G. Expression Console software (Thermo Fisher Scientific) was used for the quality assessment of microarray data. Transcriptome Analysis Console (TAC) software version 4.0.2 (Thermo Fisher Scientific) was used for background correction, normalization, and summarization of raw data (CEL files) by Robust Multichip Average plus Detection Above Background (RMA + DABG). We used the Linear Models for Microarrays (LIMMA) test with at least 1.5-fold changes (FC > 1.5) and p < 0.05 to identify differentially expressed miRNAs (DEmiRs) between samples from patients with metastatic (n = 6) or localized disease (n = 5). The TAC software constructed hierarchical clustering graphs with the DEmiRs selected for further validation. Microarray datasets are available on the Gene Expression Omnibus database (GSE172095).
5
RNA-seq Data Processing and Analysis
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Raw image data processing and normalization was performed with Transcriptome Analysis Console (TAC) software version 4.0.2 (Thermo Fisher Scientific, MA, USA) following the signal space transformation robust multi-chip analysis (SST-RMA) algorithm, which background reduced via GC Correction Version 4. Then, the gene level analysis was performed using the Limma Bioconductor package in TAC software. Filter criteria for differentially expressed genes (DEGs) were set at p-value <0.05, and −1.1> linear fold change >1.1. DEGs were analyzed using the functional annotation tools of The Database for Annotation, Visualization and Integrated Discovery (DAVID) Knowledgebase [17 (link)] (https://david.ncifcrf.gov/ . Accessed on May 2, 2022). A generic PPI network was constructed using an online tool NetworkAnalyst version 3.0 [18 (link)]. We built the first-order PPI network using the IMEx Interactome database comprised of Literature-curated comprehensive data from InnateDB [19 (link)]. Heatmaps were generated by the Morpheus online tool (https://software.broadinstitute.org/morpheus . Accessed on May 26, 2022). Gene-disease association was predicted by the Comparative Toxicogenomics Database (CTD) (http://ctdbase.org/ Accessed on July 10, 2022). Transcription factor co-occurrence was checked by Enrichr (https://maayanlab.cloud/Enrichr/ Accessed on November 5, 2022)
6
Transcriptome Analysis of Frozen Cells
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RNA extraction (QIAGEN, Hilden, Germany) of snap frozen cells were performed as described50 (link). For quality control and quantification aliquots (1 µl) were analyzed with the RNA 6000 nano Kit on a Bioanalyzer 2100 (Agilent) and Nanodrop (Thermofisher Scientific). For transcriptome analysis, 150 ng RNA was processed as previously described50 (link) and hybridized on Affymetrix Human Transcriptome Array 2.0 according to the manufacturer’s staining and scanning protocol. Data were analyzed using Expression console (Thermofisher Scientific, Darmstadt, Germany) and Transcriptome Analysis Console (TAC) software version 4.0 (Thermofisher Scientific, Darmstadt, Germany) (1.5-fold, p-value 0.05) as previously described51 (link). Full datasets are available under GSE136039 on https://www.ncbi.nlm.nih.gov/geo . Further bioinformatic analysis was performed using the knowledge-based Ingenuity Pathway Analysis (IPA) (release summer 2018 (QIAGEN, Hilden, Germany)) QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis 52 (link).
7
Microarray Data Analysis Pipeline
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CEL files generated from Affymetrix Clariom S mouse arrays were imported into the Bioconductor package oligo v1.56.0 [15 (link)] or Transcriptome Analysis Console (TAC) software version 4.0 (ThermoFisher). First, DNA microarray analyses were performed in R v4.0.3 and normalised using the Robust Multichip Average (RMA) algorithm [16 (link)]. Differential expression analysis was performed using the linear models for microarray data (limma) package v3.48.1 [17 (link)]. Linear models were determined for each transcript cluster and global variance was calculated using an empirical Bayes approach [18 (link)]. A moderated t statistic was computed for each transcript cluster with the resulting p values corrected using the Benjamini–Hochberg method to control for the false discovery rate (FDR). See ESM Methods for more details.
8
Gene expression profiling of TWIST1 knockdown
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Total RNA from TWIST1-silenced (ShTWIST) and nonsilenced cells (ShCTRL) were obtained with the RNeasy Mini kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Next, 100 ng of total RNA was used to synthesize and biotinylated cRNA according to the GeneChip whole transcription sense target labeling assay (Thermo Fisher, Waltham, MS, USA). The biotinylated cRNA was hybridized to GeneChip Human Exon 1.0 ST arrays (Thermo Fisher), washed and stained according to the manufacturer’s protocols. The GeneChip arrays were scanned using a GeneChip® Scanner 3000. Data were analyzed using Transcriptome Analysis Console (TAC) software version 4.0 (Thermo Fisher), whereby a ≥2-fold-change was used as the criteria to define upregulation or downregulation of differentially expressed genes compared to expression in ShCTRL. In silico analysis was performed using MetaCoreTM software (http://portal.genego.com/ ; accessed on May 2019) to study gene ontology, biological processes and signaling pathways of differentially expressed genes as a consequence of TWIST1 knockdown.
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