RNA was isolated from cells with the RNeasy Plus Mini Kit (Qiagen). cDNA preparation, probe labeling, hybridization, and array analysis were performed according to the manufacturer's protocols using the Affymetrix Human Genome U133A GeneChip and Affymetrix Microarray software (Affymetrix). Statistical analysis was performed with BioConductor software (http://www.bioconductor.org/ ), and data preprocessing and normalization were conducted with the Affy package (Affymetrix) as previously described (Golubovskaya et al, 2009 ).
Genechip human genome u133a
Manufactured by Thermo Fisher Scientific
The GeneChip Human Genome U133A is a high-density oligonucleotide microarray developed by Affymetrix. It is designed to analyze the expression of over 22,000 human genes and transcripts. The array provides a comprehensive view of the human transcriptome on a single chip, enabling researchers to study gene expression patterns and identify potential biomarkers or therapeutic targets.
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Lab products found in correlation
Human mapping 50 k xbai array, by Thermo Fisher Scientific (2 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Messageamp premier rna amplification kit, by Thermo Fisher Scientific (1 mentions)
Genearray scanner g2500a, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Genechips, by Thermo Fisher Scientific (1 mentions)
Bioarray highyield rna transcript labeling kit t7, by Enzo Life Sciences (1 mentions)
Human exon 1.0 st genechip, by Thermo Fisher Scientific (1 mentions)
Genearray scanner, by Thermo Fisher Scientific (1 mentions)
Genechip arrays, by Thermo Fisher Scientific (1 mentions)
14 protocols using genechip human genome u133a
1
Affymetrix Microarray Gene Expression Analysis
2
Affymetrix Gene Expression Profiling
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Total RNA was reverse transcribed to synthesis first-strand cDNA by using a T7-Oligao (dT) Promoter Primer, and then second-strand cDNA was synthesized and purified. Next, in in vitro transcription reaction, second-strand cDNA serves as a template performed for complementary RNA (cRNA) amplification and biotin labeling and was cleaned up and fragmented. All the operations were following the instructions of MessageAmp™ Premier RNA Amplification kit (Invitrogen, USA). Subsequently, the biotinylated cRNAs were hybridized to Affymetrix Human Genome U133A genechip overnight at 45°C with rocking. Genechip was then washed and stained according to the protocol of GeneChip® Hybridization, Wash, and Stain kit (Thermo Fisher Scientific, USA). Finally, Affymetrix Gene Array Scanner G2500A scanned the chip, and the scanned images were analyzed by MAS5.0 software.
3
Microarray Analysis of Sorted B-cell Populations
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Sorted B-cell subpopulations were placed in TRIzol reagent (Life Technologies) for RNA extraction following the manufacturer’s instructions. Isolated RNA was further purified with the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and processed for microarray analysis using the standard Affymetrix protocols (www.affymetrix.com ; Affymetrix, Santa Clara, CA, USA). Briefly, 1–10 μg of RNA was reverse-transcribed into complementary DNA (cDNA) (Life Technologies). The template cDNA was purified for amplification and in vitro transcription to cRNA using the BioArray™ HighYield™ RNA Transcript Labeling Kit (T7) (Enzo Life Sciences, Inc., Farmingdale, NY, USA). cRNA was biotin-labeled, purified, and hybridized to Human Genome U133A GeneChips® (Affymetrix). GeneChips® were scanned on a high-resolution scanner using GCOS version 1.2 software (Affymetrix). Data analysis was conducted after standard Affymetrix algorithm analysis (MAS5).
4
Microarray Analysis of Breast Cancer Cell Lines
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Triplicate RNA samples from MCF-7, Tam-R, and Fas-R cells were microarrayed using Affymetrix Human Genome U133A gene chips with subsequent median normalization across all datasets and log transformation using Genesifter. Heatmap profiles and log2 intensity plots were generated to visualize gene expression changes across all cell models. The genes analyzed by microarray were as follows: CD44, HMMR (the gene corresponding to the RHAMM protein), stabilin 2 (STAB-2), lymphatic vessel endothelial receptor 1 (LYVE-1), toll-like receptor 4 (TL4), intracellular adhesion molecule-1 (ICAM-1), and versican (VCAN). Statistical analysis of probe expression between cells was performed using ANOVA with Tukey post hoc analysis.
5
Robust Gene Expression Data Processing
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The raw gene expression data (CEL files) were processed and normalized using robust multi-array normalization (RMA) from R-based Bioconductor52 (link) package—affy.53 (link) Only the samples having Normalized Unscaled Standard Error (NUSE;54 (link) from R-based bioconductor52 (link)affyPLM55 package) with a median score of 1 ± 0.05 was considered high-quality arrays and selected for further analysis GSE42568.24 (link) For GSE653225 (link)–27 (link) (all samples were considered), data from two different arrays—Affymetrix GeneChip Human Genome U133A and U133B—done for the same set of samples were normalized using RMA52 (link) and merged by samples. The technical/batch effect in GSE653225 (link)–27 (link) was corrected using ComBat.56 (link) Supplementary Figure 3a shows a flow chart of the data processing and analysis for treated samples from GSE6532,25 (link)–27 (link) which also applies for untreated samples.
6
Comprehensive Gene Expression Analysis
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A comprehensive gene expression analysis was performed using an oligonucleotide microarray (GeneChip Human Genome U133A, Affymetrix, Santa Clara, CA) as described previously [38 (link)]. The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus [39 (link)] and are accessible through GEO Series accession number GSE63941 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63941 ).
7
Accessing NCI-DREAM Cancer Genomics Data
8
Affymetrix GeneChip Protocol for Gene Expression
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From an input of 250 ng RNA, cDNA targets were prepared using the Affymetrix protocol. The procedures as recommended by the manufacturer were applied to hybridize the samples to Affymetrix GeneChip Human Genome U133A plus 2 GeneChip arrays. GeneChips were washed and stained after hybridization with a fluidics station (Affymetrix) and scanned with a GeneArray scanner (Affymetrix). The samples from donor 1 exposed to 10 mM APAP did not pass quality control and were therefore excluded from further analyses.
9
Measuring mRNA Expression in Leukemic Blasts
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The mRNA expression levels obtained from leukemic blasts at diagnosis from patients in the AML02 cohort was performed using GeneChip Human Genome U133A (Affymetrix, Santa Clara, CA) and processed as described previously.23 (link) All gene expression levels were natural-log transformed before analysis. For the AML08 and AAML1031 cohort, SAMHD1 expression was obtained from RNA sequencing (RNA-seq) data. AAML1031 RNA-seq data are also available at the TARGET database (https://target-data.nci.nih.gov/Public/AML/mRNA-seq/L3/expression/ ). RPKM values were used for association analysis after log transformation.
10
Gene Expression Profiling of AML Diagnostics
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Gene expression profiling of leukemic blasts obtained at diagnosis in the AML02 discovery cohort was performed using GeneChip® Human Genome U133A [Affymetrix, Santa Clara, CA] as described previously(8 (link)). The MAS 5.0 algorithm was used to obtain normalized gene expression signals. All the gene expression data was log2 transformed before analysis. For the validation cohort, we downloaded publicly available RPKM (Reads per kilo base of transcript per million mapped reads data from TARGET database. We included 205 patients from AAML0531 and AAML03P1 clinical trials with gene expression data available from diagnostic specimens (RNAseq data from specimen obtained at relapse were not included in this analysis). We used log2(RPKM+1) values for subsequent statistical analysis, it should be noted that TARGET dataset was enriched for patients with poor outcome.
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