The non-coding RNA expression profile, GSE57969, was downloaded from the GEO database (http://www.ncbi.nlm.nih.gov/geo/ ) (20 (link)) with annotated clinical information. The GSE57969 dataset consisted of six colon tissues from six patients (five females and one male) with STC who underwent colectomy (total colectomy and ileorectal anastomosis or subtotal colectomy with antiperistaltic cecoproctostomy) and six normal colon tissues as controls from six patients (five females and one male) with colon cancer in a tumor-free area at least 5 cm away from the primary lesions. Notably, patients in the control group were free from constipation. The microarrays of all samples were scanned. The data were processed by Agilent Feature Extraction software version 9.5.3 and Gene Spring software version 11.0 (Agilent Technologies, Inc.) with normalization. The normalized data were annotated by the platform GPL11487 (Agilent-021827 Human miRNA Microarray) (17 (link)).
Agilent feature extraction software version 9
Manufactured by Agilent Technologies
Sourced in United States
Agilent Feature Extraction Software version 9.5.1 is a software tool designed to analyze data from microarray experiments. The software provides automated processing of microarray image data, including identification and quantification of features on the microarray.
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Lab products found in correlation
Microrna complete labeling and hyb kit, by Agilent Technologies (2 mentions)
Genespring gx software version 12, by Agilent Technologies (2 mentions)
Mirneasy kit, by Qiagen (2 mentions)
High resolution microarray scanner model g2505c, by Agilent Technologies (2 mentions)
Agilent microarray scanner g2565ba, by Agilent Technologies (2 mentions)
Human microrna microarray v2, by Agilent Technologies (2 mentions)
Agilent scanner g2565ca, by Agilent Technologies (2 mentions)
Agilent human 44k 60 mer oligonucleotide microarray chips, by Agilent Technologies (2 mentions)
Quick amp labeling kit, by Agilent Technologies (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Rneasy column, by Qiagen (2 mentions)
Rnalater, by Qiagen (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Gene spring software version 11, by Agilent Technologies (1 mentions)
Agilent 021827 human mirna microarray, by Agilent Technologies (1 mentions)
Salar 2, by Agilent Technologies (1 mentions)
Taqman mirna assay, by Thermo Fisher Scientific (1 mentions)
Agilent bundle, by Agilent Technologies (1 mentions)
Whole human genome microarray kit, by Agilent Technologies (1 mentions)
One color microarray based gene expression analysis quick amp labeling, by Agilent Technologies (1 mentions)
14 protocols using agilent feature extraction software version 9
1
Non-Coding RNA Expression Profile in STC
2
Microarray-based Gene Expression Profiling of Atlantic Salmon
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The current experiment used a custom designed Agilent oligonucleotide microarray platform Salar_2 (Agilent design ID: 025520), developed for Atlantic salmon and validated by two independent research groups using RT-qPCR [24 , 28 (link)–30 (link)]. The experiment consisted of 35 hybridisations (7 diets × 5 biological replicates), which were performed on nine microarray slides in a semi-randomised order (each slide with four different dietary treatments).
Each hybridisation was performed using 825 ng of Cy3-labelled experimental sample and 825 ng of Cy5-labelled common control. The aRNA was first fragmented and then hybridised at 65 °C for 17 h in an Aligent hybridization oven, as described previously [29 ]. Following hybridisation, slides were subjected to washing steps, after which they were air dried in the dark and scanned within 2 h.
Scanning was carried out at 5 μm resolution on a GenePix Personal 4100A scanner (Axon Instruments, Molecular Devices Corp., Sunnyvale, CA, USA), with the PMT values adjusted manually to ensure the mean intensity ratio of Cy3:Cy5 signal was close to one. Agilent Feature Extraction Software (version 9.5.3) was used to identify features and extract raw intensity values associated with these features for subsequent statistical analysis.
Each hybridisation was performed using 825 ng of Cy3-labelled experimental sample and 825 ng of Cy5-labelled common control. The aRNA was first fragmented and then hybridised at 65 °C for 17 h in an Aligent hybridization oven, as described previously [29 ]. Following hybridisation, slides were subjected to washing steps, after which they were air dried in the dark and scanned within 2 h.
Scanning was carried out at 5 μm resolution on a GenePix Personal 4100A scanner (Axon Instruments, Molecular Devices Corp., Sunnyvale, CA, USA), with the PMT values adjusted manually to ensure the mean intensity ratio of Cy3:Cy5 signal was close to one. Agilent Feature Extraction Software (version 9.5.3) was used to identify features and extract raw intensity values associated with these features for subsequent statistical analysis.
3
Agilent Human miRNA Microarray Profiling
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The Agilent Human miRNA Microarray Kit version 2 was used to profile miRNA expression. Total RNA (100 ng) was labeled using the Agilent miRNA Complete Labeling and Hybridization Kit (Agilent Technologies Incorporated, Santa Clara, CA, USA) according to the manufacturer's instructions. This array includes 723 mature human miRNAs based on the Sanger miRBase Release 10.1. Samples were hybridized onto an Agilent miRNA array, Human v2, at 55°C for 20 hr. After hybridization, slides were washed for 5 min in Gene Expression Wash Buffer 1 and then for 5 min in Gene Expression Wash Buffer 2, both at room temperature. Slides were scanned on an Agilent G2565A microarray scanner at 100% and 5% sensitivity settings. Agilent Feature Extraction software version 9.5.3 was used for image analysis. Arrays were scanned using an Agilent scanner and feature-extracted using Agilent Feature Extraction Software, version 10.5.1.1. TaqMan miRNA assays were used to quantify the levels of mature miRNAs following the manufacturer's protocol (Applied Biosystems).
4
Microarray-based Gene Expression Profile
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In order to determine gene expression profiles, 4 × 44K v.2 DNA microarrrays (whole human genome microarray kit, Agilent technologies, cat no. G4845A, Santa Clara, CA, USA) were used. The procedures for hybridization using the fluorescent dye Cy3 followed the manufacturer’s protocols (one-color microarray-based gene expression analysis—low input quick amp labeling). The images were captured by the reader Agilent bundle, according to the parameters recommended for bio-arrays and extracted by Agilent feature extraction software version 9.5.3. Spots with two or more flags (low intensity, saturation, controls, etc.) were considered as NA, that is, without valid expression value. An in-house algorithm in R environment (version 3.4.4 [13 ]) was used for excluding transcript spots presenting one or more NAs and for converting gene expression values to log base 2. Through this procedure, we identified 6876 or 7555 valid gene ontology (GO) annotated genes for Caco-2/EH41 or Caco-2/Ec472/01 groups, respectively. Data normalization was performed using limma package [14 ] in R environment (version 3.4.4 [13 ]). All microarray raw data have been deposited in expression omnibus (GEO) public database under accession number GSE104492.
5
MELK Pathway Profiling in Glioma Cells
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Initially we performed a microarray analysis to select genes involved in MELK pathway. The differential gene expression profiles in the U87MG cell line transfected with MELKsiRNA compared with NTC-transfected cells was analyzed by 44 K DNA microarrrays (Whole Human Genome Microarray Kit, Agilent Technologies, Santa Clara, CA).
The human U87MG glioma cell lines transfected (1x105 cells) with oligonucleotide MELK siRNA reduced MELK expression levels by 95 and 96 % in first and second independent experiments, respectively, at the 48 h confirmed by qRT-PCR and western blotting.
Hybridisation was performed according to the protocol provided by the manufacturer (One-Color Microarray-Based Gene Expression Analysis—Quick Amp Labeling, Agilent Technologies). The images were captured by the reader Agilent Bundle according to the parameters recommended for bioarrays and extracted using Agilent Feature Extraction software version 9.5.3, considering spots with none or only one flag. The selected transcripts were analyzed with the R software version 2.11.0 (R Development Core Team, 2008) and the Lowess test was applied for array normalisation. We obtained common genes with reduced expression when MELK was silenced compared with the control considering a fold change (NTC/MELK) ≥2 in two independent assays. These genes were annotated using WebGestalt [49 (link)].
The human U87MG glioma cell lines transfected (1x105 cells) with oligonucleotide MELK siRNA reduced MELK expression levels by 95 and 96 % in first and second independent experiments, respectively, at the 48 h confirmed by qRT-PCR and western blotting.
Hybridisation was performed according to the protocol provided by the manufacturer (One-Color Microarray-Based Gene Expression Analysis—Quick Amp Labeling, Agilent Technologies). The images were captured by the reader Agilent Bundle according to the parameters recommended for bioarrays and extracted using Agilent Feature Extraction software version 9.5.3, considering spots with none or only one flag. The selected transcripts were analyzed with the R software version 2.11.0 (R Development Core Team, 2008) and the Lowess test was applied for array normalisation. We obtained common genes with reduced expression when MELK was silenced compared with the control considering a fold change (NTC/MELK) ≥2 in two independent assays. These genes were annotated using WebGestalt [49 (link)].
6
Microarray Analysis of Mouse miRNA
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Total RNA was isolated using a miRNeasy Kit (Qiagen, Valencia, California, USA) according to the manufacturer’s instructions. Quality-confirmed total RNA samples were determined with the Quant-iT™ RNA Assay kit (Invitrogen, Carlsbad, California, USA). The samples were labeled with cyanine 3-pCp and subsequently hybridized to the Agilent mouse microRNA microarray release version 15 (1881 mouse miRNAs represented) using a microRNA Complete Labeling and Hyb Kit (Agilent Technologies) for 20 h. After washing, the slides were scanned with a G2565BA scanner, and the data were analyzed and monitored with Agilent Feature Extraction Software version 9.5.1 and GeneSpring GX software version 12.5 (Agilent Technologies).
7
Mouse miRNA Expression Profiling
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Total RNA was isolated using a miRNeasy kit (Qiagen). After dephosphorylation and denaturation, the total RNA was labeled with cyanine 3-pCp and subsequently hybridized to an Agilent mouse microRNA microarray (release version 15) using the microRNA Complete Labeling and Hyb Kit (Agilent Technologies, Inc.). After hybridization for 20 h, the slides were washed using the Gene Expression Wash Buffer (Agilent Technologies, Inc.), scanned using an Agilent Scanner G2565BA, and processed and analyzed using Agilent Feature Extraction Software version 9.5.1. The raw data were analyzed using GeneSpring GX software version 12.5 (Agilent Technologies, Inc.).
8
Microarray Analysis of RDX Exposure
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An Axon GenePix® 4000B Microarray Scanner (Molecular Devices Inc., Sunnyvale, CA) was used to scan microarrays at 5 μm resolution. Data were extracted from microarray images using Agilent Feature Extraction software, version 9.5.1 (Agilent Technologies). Microarray data were normalized to the 50th percentile within each array followed by median scaling among all exposures using GeneSpring Software version 7.3 (Agilent Technologies). GeneSpring was additionally used to conduct statistical analyses to identify differentially expressed transcripts (DET) among treatments using one-way ANOVA including Benjamini and Hochberg multiple testing corrections [33 ]. A post-hoc test including a parametric t-test (p = 0.05) and log2 fold change cutoff of ≥ 1.5 was used to discern statistically significant differences in transcript expression for each RDX treatment relative to the control. Given the sense-antisense architecture of the microarray (see above), all differentially expressed microarray targets were examined to identify and eliminate microarray targets for which both the sense and antisense probes were differentially expressed. Microarray data are publicly available at the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/info/linking.html ) under series accession GSE27624.
9
Microarray Data Analysis Protocol
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An Agilent Technologies, High-Resolution Microarray Scanner (Model G2505C, Agilent Technologies, Santa Clara, CA, USA) was used to scan microarray images at 2 μm resolution. Data were extracted using Agilent Feature Extraction software, version 9.5.1 (Agilent Technologies). Internal control spots were analyzed to confirm that signal data was within the linear range of detection. GeneSpring version GX 11.0.2 (Agilent Technologies) was used to normalize data using quantile normalization followed by baseline transformation to the median of all samples. Statistical analysis was performed using GeneSpring where two-way ANOVA (p ≤ 0.05) with Benjamini-Hochberg multiple-testing correction and 1.5 fold-change cutoff was used to identify differentially expressed transcripts. Principal components analysis and hierarchical clustering analysis for transcripts with significant differential expression were conducted using GeneSpring and Multi-Experiment Viewer software version 4.9 [23 (link)], respectively.
10
High-Resolution Microarray Data Analysis
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An Agilent Technologies, High‐Resolution Microarray Scanner (Model G2505C, Agilent Technologies) was used to scan microarray images at 2‐μm resolution. Data were extracted from microarray images using agilent feature extraction software, version 9.5.1 (Agilent Technologies). Microarray data were normalized to the 75th percentile within each array followed by median scaling among all exposures using genespring Software version GX12.5 (Agilent Technologies). Methods for the statistical analysis of microarray data are provided in Section 2.8 .
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