CGH analysis was performed using our custom Agilent microarray (4×44K format)12 (link). Genomic DNA from wild type or mutants was digested with AluI and RsaI. After complete digestion, mutant DNA was labeled with Cy-5 dCTP (Amersham Biosciences) and wild type DNA was labeled with Cy-3 dCTP (Amersham Biosciences) using the BioPrime® Array CGH Genomic Labeling kit (Invitrogen). Equal amounts of labeled DNA (1.5 ug) were competitively hybridized onto the microarray. Prehybridization, probe hybridization, washing, and drying steps for arrays were performed as for ChIP-chip experiments12 (link). Arrays were scanned using an Agilent scanner (Agilent) and analyzed using Agilent Feature Extraction (Agilent). Signal intensity ratios between Cy5 (mutant) and Cy3 (Wild type) were calculated from rProcessedSignal and gProcessedSignal values according to Agilent Feature Extraction. The log2 transformed Cy5/Cy3 ratio is plotted along the chromosome.
Agilent feature extraction
Manufactured by Agilent Technologies
Sourced in China, United States
Agilent Feature Extraction is a software application designed to analyze microarray data. It enables users to extract meaningful information from complex gene expression data, facilitating the identification and quantification of genetic features.
Automatically generated - may contain errors
Lab products found in correlation
Agilent microarray scanner, by Agilent Technologies (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Agilent gene expression hybridization kit, by Agilent Technologies (3 mentions)
Genespring gx v12, by Agilent Technologies (2 mentions)
Agilent scanner, by Agilent Technologies (1 mentions)
Cy5 dctp, by Cytiva (1 mentions)
Cy3 dctp, by Cytiva (1 mentions)
Bioprime array cgh genomic labeling kit, by Thermo Fisher Scientific (1 mentions)
Oligonucleotide array based cgh for genomic dna analysis, by Agilent Technologies (1 mentions)
Sureprint g3 human cgh microarray, by Agilent Technologies (1 mentions)
Dna microarray scanner, by Agilent Technologies (1 mentions)
Agilent microarray scanner agilent p n g2565ba, by Agilent Technologies (1 mentions)
Quick amp labeling kit, by Agilent Technologies (1 mentions)
Whole human genome oligo microarray, by Agilent Technologies (1 mentions)
R software package, by R Project for Statistical Computing (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Lncrna mrna human gene expression microarray v4, by CapitalBio (1 mentions)
Agilent genespring, by Agilent Technologies (1 mentions)
Genespring software v13, by Agilent Technologies (1 mentions)
19 protocols using agilent feature extraction
1
Comparative Genomic Hybridization Analysis
2
Array CGH Analysis of Human Genomes
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Array CGH was performed using the Agilent Technologies Platform (Santa Clara, CA, USA) with a SurePrint G3 Human CGH Microarray harboring 180,000 oligonucleotide probes.
Labeling, purification, and hybridization of DNA samples were carried out according to the manufacturer’s protocol (Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis, version 7.3). Slides were scanned using a DNA Microarray Scanner (Agilent Technologies), and TIFF images were obtained from Agilent Scan Control software. Raw data were generated using Agilent Feature extraction and analyzed by Agilent Cytogenomics 3.0. Copy number variation analysis was performed using the ADAM2 algorithm. The aberration filter was set to detect a minimum number of three consecutive probes/region, and the minimum absolute average Log Ratio (MAALR) was ±0.25.
Labeling, purification, and hybridization of DNA samples were carried out according to the manufacturer’s protocol (Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis, version 7.3). Slides were scanned using a DNA Microarray Scanner (Agilent Technologies), and TIFF images were obtained from Agilent Scan Control software. Raw data were generated using Agilent Feature extraction and analyzed by Agilent Cytogenomics 3.0. Copy number variation analysis was performed using the ADAM2 algorithm. The aberration filter was set to detect a minimum number of three consecutive probes/region, and the minimum absolute average Log Ratio (MAALR) was ±0.25.
3
Profiling Transcriptome Alterations in CRC Metastasis
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Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The RNA quality was confirmed by formaldehyde agarose gel electrophoresis and quantified by NanoDrop ND-1000. The samples (CRC tissues with or without liver metastasis, Table S1 ) were used to synthesize double-stranded cDNA, and the cDNA was then labeled and hybridized to the LncRNA Expression Microarray (Arraystar 8x60Kv3.0, Rockville, USA) according to the manufacturer's protocol. After hybridization, the arrays were washed, and the slides were scanned with an Agilent Microarray Scanner (Agilent p/n G2565BA). Raw data were extracted as pair files using the Agilent Feature Extraction. The random variance model was used to identify the differentially expressed genes. The paired t-test was used to calculate the P-value. The threshold set for up- and down-regulated genes was a fold change >=4.0 and a P-value <= 0.05, respectively. The microarray data have been deposited into the NCBI Gene Expression Omnibus (GEO) database and are available through GEO with the accession number GSE95423.
4
Total RNA Extraction and Microarray Analysis
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Total RNA was extracted from mPFC tissues using TRIzol, and the RNA was reverse transcribed into cDNA (Takara, Dalian, China). Then, cDNA was transcribed into cRNA labelled with Cyanine‐3‐CTP (Cy3) (Agilent, USA). The microarray was scanned using an Agilent Microarray Scanner (Agilent, USA) after fragmentation, hybridization, and washing. RNAs with differential expression were identified using a whole genome microarray (fold change > 2, p < 0.05). Microarray analysis was performed using Agilent Feature Extraction by Oebiotech (China).
5
Differential Expression of lncRNAs in Primary Microglia
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Total RNA of the cells was extracted by using a TRIzol commercial kit (Invitrogen, USA). And Quick Amp Labeling Kit, One-Color (Agilent, USA) was used to prepare labeling reaction. Then labeled/amplified RNA and labeled cRNA QC were purified by RNeasy Mini Kit (Qiagen, German). After fragmentation, hybridization, and microarray wash, the microarray was scanned by Agilent Microarray Scanner (Agilent, USA). LncRNAs with differential expressions in primary microglial cells were picked out by the whole genome microarray expression profiling with the fold change > 2 and adjusted P < 0.05. The microarray analysis was performed with Agilent Feature Extraction by Oebiotech, Shanghai, China. In addition, sample preparation and microarray hybridization were performed according to the manufacturer’s standard protocol with only minor modifications.
6
Microarray Analysis of Gene Expression
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Quick Amp Labeling Kit (Agilent p/n 5190-0442, Santa Clara, CA, USA) and RNeasy Mini Kit (Qiagen p/n 74104) were used for labeling of mRNA and cRNA, respectively. Agilent Gene Expression Hybridization Kit (Agilent p/n 5188-5242) was used for mRNA hybridization. Agilent Microarray Scanner (Agilent p/n G2565BA) was used to read microarrays.
Data were extracted with Agilent Feature Extraction (Agilent). With Multi-Class Dif analysis, P < 0.05 and false discovery rate < 0.05, significant differentially expressed mRNAs were obtained. Data were calculated with Serial Test Cluster (Ernst et al., 2005). Among 80 default patterns, P < 0.05 was used to obtain dynamic expression patterns of differential genes. Figures were used to quantify the dynamic changes.
Data were extracted with Agilent Feature Extraction (Agilent). With Multi-Class Dif analysis, P < 0.05 and false discovery rate < 0.05, significant differentially expressed mRNAs were obtained. Data were calculated with Serial Test Cluster (Ernst et al., 2005). Among 80 default patterns, P < 0.05 was used to obtain dynamic expression patterns of differential genes. Figures were used to quantify the dynamic changes.
7
Circular RNA Microarray Analysis
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Scanned images were imported into Agilent Feature Extraction software version 11.0.1.1 (Agilent Technologies, Inc.) for raw data extraction. Quantile normalization of raw data and subsequent data processing were performed using the R software package (www.arraystar.com/arraystar-human-circular-rna-microarray ). Following quantile normalization of the raw data, low-intensity filtering was performed and circRNAs having the ‘P’ or ‘M’ flags (‘All Targets Value’) in ≥3 out of 6 samples were retained for further analyses. Subsequently, samples were clustered hierarchically with Cluster software version 2.0 (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm ) to evaluate the robustness of the formed clusters, using the correlation-centered metric and average linkage-clustering algorithm. When comparing profile differences between the GC and control groups, the fold-change between the groups for each circRNA was computed. The statistical significance of each difference was estimated by Student's t-test. CircRNAs exhibiting a ≥2-fold change in expression (P<0.05) were considered to be significantly differentially expressed.
8
Microarray Analysis of Lung Tissue RNA
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Lung tissue RNA was isolated using Trizol reagent (Life Technologies Corporation, CA, USA), and RNA quality was determined. RNA labeling reaction was performed using a Quick Amp Labeling Kit, One-Color (p/n 5190–0442, Agilent Technologies, CA, USA) and purified by RNeasy Mini Kit (p/n 74,104, Qiagen, Hilden, Germany). Then, labeled/amplified cRNA was hybridized using Agilent Gene Expression Hybridization Kit (p/n 5188–5242, Agilent), and then the microarray was washed. We used Agilent Microarray Scanner (p/n G2565BA, Agilent) and Agilent Feature Extraction to scan and extract data. (Aksomics Inc., Shanghai, China)
9
Transcriptional Profiling of CD161+ NK Cells
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CD161+CD161+ or CD161− NK cells (singlet, alive, CD14−CD19−CD3−CD56+) were sorted using a MoFlo MLS cell sorter (Beckman Coulter) from four donors. Purity was >96%. Three out of four donors were CMV seronegative, while the seropositivity of the remaining donor is unknown. Cell pellets were snap frozen and sent to Miltenyi Biotec Genomic Services (Bergisch Gladbach) for RNA extraction and hybridization to Agilent Whole Human Genome Oligo Microarray. Raw microarray image files were processed using Agilent feature extraction, and differential gene expression was analyzed using the Rosetta Resolver gene expression data analysis system (Rosetta Biosoftware). Hierarchical clustering of differentially regulated genes (>2-fold, p < 0.01) was completed using the heatmap function in GENE-E (Broad Institute). The NCBI Gene Expression Omnibus accession number for the microarray data reported in this paper is GSE98702. The NK cell data were compared to genes significantly upregulated (>2-fold, p < 0.05) in a previously published dataset of CD161intermediate(int) CD8+ T cells compared to the CD161−CD8+T cells (25 (link), 27 (link), 28 (link)) using gene set enrichment analysis (GSEA) v2.1.0 (29 (link)).
10
Differential Expression Analysis of circRNAs
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Scanned images were imported into Agilent Feature Extraction software version 11.0.1.1 (Agilent Technologies, Inc.) for raw data extraction. Quantile normalization of raw data and subsequent data processing were performed using the R software package (R Project for Statistical Computing, Vienna, Austria). After quantile normalization of the raw data, low intensity filtering was performed, and the circRNAs that were flagged as ‘P’ or ‘M’ (‘All Targets Value’) in at least 3 out of 6 samples were retained for further analyses. When comparing profile differences between two groups (including disease vs. control), the ‘fold change’ (i.e., the ratio of the group mean averages) between the groups for each circRNA was computed. The statistical significance of the difference was determined by a t-test. circRNAs with fold changes >1.5 and P<0.05 were selected as significantly differentially expressed circRNAs. circRNA sequences were predicted by bioinformatics methods, as described previously (6 (link)).
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