The Human Transcriptome 2.0 Array (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to perform the whole transcriptome analysis as described previously [30 (link)]. Quality control was performed in Transcriptome Analysis Console (TAC) 4.0 (Applied Biosystems, Waltham, MA, USA) as per manufacturer’s instructions using the probe-level intensity files. The Signal Space Transformation in conjunction with the Robust Multiarray Average method (SST-RMA) were used to perform data normalization. Batch normalization was performed using ComBat method [31 (link)] to adjust for batch effects between the cohorts since the data were collected during different time intervals. For further analysis, coding genes were selected.
Human transcriptome 2.0 array
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Human Transcriptome 2.0 array is a high-density microarray designed for comprehensive analysis of the human transcriptome. It provides coverage of over 70,000 well-annotated human genes and transcripts, enabling researchers to study gene expression profiles across the entire human genome.
Automatically generated - may contain errors
Lab products found in correlation
Transcriptome analysis console tac 4, by Thermo Fisher Scientific (1 mentions)
Transcriptome analysis console software version 4, by Thermo Fisher Scientific (1 mentions)
Wt expression kit, by Thermo Fisher Scientific (1 mentions)
Genechip microarray, by Thermo Fisher Scientific (1 mentions)
Human genome u133a 2.0 array, by Thermo Fisher Scientific (1 mentions)
Matlab, by MathWorks (1 mentions)
Mouse gene 1.0 st array, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
8 protocols using human transcriptome 2.0 array
1
Whole Transcriptome Analysis with Human Transcriptome 2.0 Array
2
Whole Transcriptome Analysis Using Human Transcriptome 2.0 Array
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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).
3
Transcriptome Analysis of Paired Samples
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Total RNA (250 ng per sample) was processed using the Ambion WT expression kit (Austin, TX, United States) and labeled with Affymetrix GeneChip (Santa Clara, CA, United States) whole-transcript sense target labeling assay, followed by hybridization to the Affymetrix Human Transcriptome 2.0 array according to the manufacturer’s protocols. Following hybridization and scanning, quality control and robust multichip averaging were performed on the feature intensity files using the Affymetrix Expression Console software version 1.4. Gene-level differential expression analysis was subsequently performed using the Affymetrix Transcriptome Analysis Console software version 3.0 using the paired-sample analytical pipeline (one-way repeated-measure ANOVA), and the Benjamini–Hochberg false discovery rate (FDR)-controlling procedure (Reiner et al., 2003 (link)). For the default differential gene expression analysis, a linear fold-change threshold of ± 2 and an FDR of 10% was applied.
4
Panobinostat's Impact on Gene Expression
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The effect of panobinostat on gene expression was evaluated using an Affymetrix Human Transcriptome 2.0 array (42 ). Briefly, using PAXgene blood RNA tubes (PreAnalytiX), five million PBMCs were directly lysed following isolation. Samples were stored at −80°C. RNA isolation, RNA integrity verification, hybridization to the Affymetrix Human Transcriptome 2.0 array (70,523 probes), and realigning were performed (Aros Applied Biotechnology, Aarhus, Denmark). Gene expression was evaluated at baseline (visit 2), day 4 on-panobinostat (visit 4), and follow-up (visit 12) for all patients. Gene expression data were normalized using Robust Multi-Array Average (RMA) algorithms (43 (link)).
5
Identifying MELK Downstream Targets
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To identify the downstream targets of MELK, we performed Affymetrix Genechip microarray using parental, Cas9-p15 control, and two MELK KO (C3 and C28) MDA-MB-231 cells. In brief, the cells (1 × 106 cells) were cultured in 10-cm plates for 48 hours, followed by RNA extraction using the Invitrogen PureLink RNA Mini Kit (Thermo Fisher Scientific). RNA expression was measured using Human Transcriptome 2.0 Array (Affymetrix Inc) at the Sequencing and Microarray Facility at MD Anderson according to standard Affymetrix protocols. Differential gene expression profile analysis was performed (see Supplementary Methods and Materials for details).
6
Bioinformatics Analysis of Diabetic Kidney Disease
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The chip data used for bioinformatics analysis was downloaded from the online NCBI Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/ ). We used “diabetic kidney disease,” “diabetic nephropathy,” and “Homo sapiens” as search terms to retrieve datasets. After careful screening, the independent chip dataset GSE96804 was selected as the training set for subsequent analysis.[18 (link)] The dataset GSE96804 consisted a total of 61 human glomeruli tissue samples. The glomeruli samples were from 41 DKD patients (DKD group) and 20 healthy controls (Control group). In addition, 2 more human chip datasets GSE104948 and GSE30122 in both glomerular and tubular tissues were used as the validation sets.[19 (link),20 (link)] The detection platforms used in this study were Affymetrix Human Transcriptome 2.0 Array and Affymetrix Human Genome U133A 2.0 Array for the training and validation sets respectively. Ethical approval was unnecessary in this study because the chip data was downloaded from GEO database and we do not conduct new experiments in patients or animals.
7
Gene Expression Signatures for TLS Identification
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The presence of TLSs could be evaluated by a 12-gene expression signature, as previously described, including CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21, CXCL9, CXCL10, CXCL11 and CXCL13.24 (link) We retrieved the gene expression profile data including 171 CRCLM samples from Gene Expression Omnibus (accession number GSE159216), analyzed on Affymetrix Human Transcriptome 2.0 Arrays. Clinical annotations have been downloaded for further analysis. For each sample, we calculated the geometric mean of the above 12 genes. Subsequently, sample with a geometric mean superior to the third quartile were classified as TLS+, while sample with a geometric mean inferior to the third quartile were classified as TLS-. The associations between TLS positivity with molecular subtype and clinical outcome were analyzed.
8
Transcriptome Profiling of Mouse and Human Cells
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Total mRNA was extracted using the QIAGEN RNeasy kit (QIAGEN) from 9 mouse GC cultures, 3 mouse NSC cultures, and 61 human GBM cell lines. The RNA was labeled and hybridized on Affymetrix Mouse Gene ST 1.0 arrays (mouse samples) or Affymetrix Human Transcriptome 2.0 arrays (human samples) at the SciLifeLab Array and Analysis Facility. The raw data were normalized using the Affymetrix Expression Console software (http://www.affymetrix. com/estore) using the robust multi-array average method (Li and Wong, 2001) . All subsequent data processing and analyses were carried out in R using packages available from the Bioconductor project (https://www.bioconductor.org) and MATLAB (version R2014b; The MathWorks).
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