RNA was extracted using Trizol Reagent (Life Technologies, Inc, New York, USA) according to the manufacturer’s protocol. Total RNA was purified with RNeasy Plus Micro Kit (Qiagen, Valencia, USA) to remove genomic DNA. The RNA integrity number (RIN) was measured with Agilent RNA 6000 NanoChips on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). The quantity was measured with a NanoDrop 1000 (NanoDrop Technologies, Inc, Wilmington, USA). A total of 300 ng of each RNA sample with RIN higher than 9 was labeled with GeneChip Whole Transcript (WT) Sense Target Labeling Assay (Affymetrix, Santa Clara, USA) and hybridized to Affymetrix GeneChip Human Gene 1.0 ST Arrays at 45 °C for 16 h. Arrays were stained and washed on Affymetrix GeneChip Fluidics 450 following the manufacturer’s protocol and scanned with an Affymetrix GeneChip Scanner 3000 7G System. Array data files were generated with GeneChip® Command Console® Software (AGCC) (Affymetrix, Santa Clara, USA) (ArrayExpress accession ID# E-MTAB-11492) and statistical analysis was carried out on software of GeneSpringTM (Agilent Technologies). The fold change between normal and OA samples was based on the p < 0.05 from a t-test (Asymptotic and Benjamini Hochberg FDR).
Genechip command console software agcc
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneChip® Command Console® Software (AGCC) is a software suite designed to control and manage Affymetrix GeneChip® microarray instruments. It provides the core functionality to operate and analyze data from Affymetrix GeneChip® systems.
Automatically generated - may contain errors
Lab products found in correlation
Fluidics station 450, by Thermo Fisher Scientific (3 mentions)
Genechip human gene 1.0 st array, by Thermo Fisher Scientific (3 mentions)
Genechip fluidics station 450, by Thermo Fisher Scientific (3 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Genechip scanner 3000 7g system, by Thermo Fisher Scientific (1 mentions)
Agilent rna 6000 nano chip, by Agilent Technologies (1 mentions)
Genechip whole transcript sense target labeling assay, by Thermo Fisher Scientific (1 mentions)
Genechip fluidics 450, by Thermo Fisher Scientific (1 mentions)
Genespring, by Agilent Technologies (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
Rneasy plus micro kit, by Qiagen (1 mentions)
Encore biotin module, by Tecan (1 mentions)
Genearray scanner 3000 7g, by Thermo Fisher Scientific (1 mentions)
Human gene 2.0 st array, by Thermo Fisher Scientific (1 mentions)
Gene array 3000 scanner, by Thermo Fisher Scientific (1 mentions)
Wt plus reagent kit, by Thermo Fisher Scientific (1 mentions)
Expression console software, by Thermo Fisher Scientific (1 mentions)
Ambion wt expression kit, by Thermo Fisher Scientific (1 mentions)
Genechip wt terminal labeling kit, by Thermo Fisher Scientific (1 mentions)
12 protocols using genechip command console software agcc
1
RNA Extraction and Microarray Analysis of Osteoarthritis
2
Pico Amplification and Microarray Analysis
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RNA was amplified and labeled using a pico amplification kit according to manufactures instructions. In short, approximately half of the total RNA from each sample (5 μl of 10 μl) was amplified using the Ovation Pico WTA v.2 RNA Amplification System from NuGEN® Inc. (NuGEN®, San Carlos, CA, USA) and biotin labeling was performed with the Encore Biotin Module (NuGEN®). The labeled samples were hybridized to the Human Gene 1.0 ST GeneChip array (Affymetrix, Santa Clara, CA, USA). The arrays were washed and stained with phycoerytrin conjugated streptavidin (SAPE) using the Affymetrix Fluidics Station® 450, and the arrays were scanned in the Affymetrix GeneArray® 3000 7G scanner to generate fluorescent images, as described in the Affymetrix GeneChip® protocol. Cell intensity files (CEL files) were generated in the GeneChip® Command Console® Software (AGCC) (Affymetrix, Santa Clara, CA, USA).
All samples are MIAME compliant and were handled according to SOP in the Microarray Center. A total of 27 CC and 19 MGC samples were amplified and hybridized to arrays. The 46 samples were submitted to ArrayExpress at EMBL using MIAMExpress. The experiment accession number is E-MTAB-4012.
All samples are MIAME compliant and were handled according to SOP in the Microarray Center. A total of 27 CC and 19 MGC samples were amplified and hybridized to arrays. The 46 samples were submitted to ArrayExpress at EMBL using MIAMExpress. The experiment accession number is E-MTAB-4012.
3
Whole Blood RNA Profiling via Microarray
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RNA was extracted from whole blood collected in PAXgene tubes with the PAXgene miRNA Blood kit (PreAnalytiX, Qiagen) in the fasting state. RNA integrity and concentration were analyzed on a 2100 Bioanalyzer (Agilent Technologies, DK). A minimum of 450 ng of total RNA with mean RIN values of 8.9 was used as input. RNA was amplified and labelled using the WT PLUS reagent kit (Thermo Fisher Scientific, Carlsbad, CA, USA). The labelled samples were hybridized to the Human Gene 2.0 ST array (Affymetrix, Santa Clara, CA, USA). The arrays were washed and stained with phycoerytrin-conjugated streptavidin using the Affymetrix Fluidics Station® 450, and the arrays were scanned in the Affymetrix GeneArray® 3000 scanner to generate fluorescent images, as described in the Affymetrix GeneChip® protocol. Cell intensity files (CEL files) were generated in the GeneChip® Command Console® Software (AGCC) (Affymetrix, USA). The microarray data were modelled using the RMA (Robust Multichip Average) approach, followed by mean one step probe set summarization giving each gene a single expression value, all done using the software package Partek Genomics Suite 6.
4
Affymetrix miRNA Expression Analysis
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Raw data were extracted automatically in Affymetrix data extraction protocol using the software provided by Affymetrix GeneChip Command Console Software (AGCC) (Affymetrix). The CEL files import, miRNA level RMA + DABG-All analysis and result export using Affymetrix Expression Console Software. Array data were filtered by probes annotated species. The comparative analysis between test sample and control sample was carried out using fold-change and independent T test in which the null hypothesis was that no difference existed between the 2 groups. The miRNAs not detected in any samples were excluded from analysis. miRNAs not expressed completely in either group were also excluded. Significance of differential expression between two groups was estimated by t test for those miRNAs of at least a 1.5-fold reduced or increased mean expression between the 2 groups. All statistical tests of differentially expressed (DE) genes were conducted using the R statistical language v. 3.2.2. of the software.
5
Gene Expression Profiling of Cells
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To compare the gene expression profiles of the different cells isolated, an Affymetrix Gene Chip Human Gene 1.0 ST Array (Affymetrix, Santa Clara, CA, USA) was used. 150 ng of total ribonucleic acid (RNA) was subjected to an Ambion WT Expression Kit (Thermo Fisher Scientific, Waltham, MA, USA) and a GeneChip WT Terminal Labeling Kit (Affymetrix) according to the manufacturers’ protocol, then washed and stained on FS-450 fluidics station (Affymetrix). The signal intensities were detected by Hewlett Packard Gene Array Scanner 3000 7 G (Hewlett Packard, Palo Alto, CA, USA). The scanned images were processed using GeneChip Command Console Software (AGCC) (Affymetrix) and the CEL files were imported into GeneSpring GX 12.6 software (Agilent Technologies Inc, Santa Clara, CA, USA). Robust microarray analysis (RMA) was applied for normalization. Based on the literature, stem cells-related genes were selected and statistical analysis was performed (One-way ANOVA with Tukey post hoc test and Benjamini-Hochberg FDR; fold change cut off being set at 2) to calculate p values and fold change.
6
RNA Extraction and Microarray Analysis
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RNA extraction was prepared with the RNeasy plus mini kit (Qiagen, Germany) for microarray analysis. Amplification and labeling of total RNA were performed using GeneChip® 3’ IVT Express Kit following manufacture’s protocol (Affymetrix 2004, CA, USA). Biotin-labeled target samples were hybridized to the GeneChip® Clariom_S_Human_HT containing probes for over 18k genes. Target hybridization was processed on the GeneTitan® Instrument according to manufacturer’s instructions provided for Expression Array Plates (P/N 702933). Images were analyzed using the GeneChip® Command Console Software (AGCC) (Affymetrix, CA, USA). Microarray data were processed using the statistical computing R-program (R version 3.4.2) and Bioconductor tools [38 (link)]. The gene expression values were normalized using Robust Multi-array Average (RMA) [39 (link)]. Individual probes were grouped into gene-specific probe sets based on Entrez Gene using the metadata package clariomshumanhthsentrezg (version 22.0.0) [40 (link)].
7
Microarray Analysis of Gene Expression
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Microarray analysis was performed using Affymetrix GeneChip Human Gene U133A 2.0 Array (HG-U133A) (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. Briefly, isolated RNA (100 ng) was reverse transcribed to double-stranded cDNA. In vitro transcription synthesis of aRNA was performed at 40°C during 16 h. In all cases, 12 μg of each aRNA preparation was fragmented and hybridized onto a GeneChip Human Gene U133A 2.0 Array for 16 hours in a Hybridization Oven 640 (Affymetrix). The arrays then were washed and stained on a GeneChip Fluidics Station 450 (Affymetrix). Scanned images were converted to CEL files by GeneChip Command Console Software (AGCC) (Affymetrix). Raw expression values were pre-processed using the Robust Multiarray Averaging method. These normalized values were used for all subsequent analyses. Data were subjected to non-specific filtering to remove low signal and low variability genes. The selection of differentially expressed genes was based on a linear model analysis with empirical Bayes modification for the variance estimates. p-values were adjusted to obtain a strong control over the false discovery rate using the Benjamini-Hochberg method.
8
Profiling Memory B-cell Transcriptome
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Stored fluorescence-activated cell sorted (FACS) cells from normal Thy and BM were analyzed on Human Exon 1.0 ST (Exon) Arrays (Affymetrix, Santa Clara, CA) following a method for RNA isolation and amplification as described previously by our group [31] . Array data ''.CEL'' files from the Exon array were generated by the Affymetrix GeneChip Command Console Software (AGCC). The preprocessing of data was performed in Partek Genomics Suite version 6.5 (Partek, St. Louis, MO). The configuration consisted of probe filtering by including only core Meta-Probeset, adjustment for GC content, RMA background correction, and quartile normalization. The Affymetrix library file HuEx-1_0-st-v2.r2.dt1.hg18.core.mps was used. For genelevel analysis, exons for each gene were summarized to gene expression values using one-step Turkey's biweight algorithm. Significantly upregulated memory B-cell genes were identified by a two-way ANOVA model, including donor as random effect and B-cell subsets as fixed effect. Gene lists were generated by identifying significantly expressed genes between Thy memory IgG B cells and BM memory B cells with a fold change of 2 and a p value !0.05. Gene lists containing upregulated genes were subsequently created by only including genes with a fold change O2.
9
Affymetrix GeneChip miRNA 4.0 Array Processing
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The Affymetrix GeneChip miRNA 4.0 array process was carried out according to the manufacturer’s protocol (Affymetrix, Santa Clara, CA, USA). Approximately 1,000 ng of RNA was labeled with the FlashTag Biotin RNA Labeling Kit (Genisphere, Hatfield, PA, USA). The labeled RNA was then quantified, fractionated, and hybridized to the miRNA microarray chip according to the standard procedures recommended by the manufacturer. The labeled RNA was heated to 99°C for 5 minutes and then to 45°C for another 5 minutes. RNA-array hybridization was performed in an Affymetrix Fluidics Station 450 with agitation at 60 rotations per minute for 16 hours at 48°C. The chips were washed and stained using a GeneChip Fluidics Station 450 (Affymetrix) and then scanned with an Affymetrix GCS 3000 scanner. Signals were quantified using the Affymetrix GeneChip Command Console (AGCC) software.
10
Transcriptional Regulation of HIF-1α and HIF-2α
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SW1353 cells were transfected with the control, HIF1A, or EPAS1 siRNAs. Three biological replicates were used for each group. cDNA was synthesized using the GeneChip Whole Transcript Amplification kit (Affymetrix) as described by the manufacturer. Microarray service was provided by Macrogen Inc. and performed using the GeneChip® Human Gene 2.0 ST Array (Affymetrix). Array data export processing and analysis were performed using the Affymetrix® GeneChip Command Console® (AGCC) Software. To determine differentially expressed genes between comparison samples, |log2(fold change)| > 0.263 and P-value (paired t test)< 0.05 were used as cut-offs. Publicly available HIF-2α ChIP-Seq datasets (GSM3417828, GSM3417842)30 were analyzed using the algorithms STAR67 (link) and HOMER68 (link) for reads alignment, peak calling, and motif filtering.
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