Total RNA was extracted from differentiating cells either treated or not treated (control samples) with single dose of TGF-β1 at D5 of induction using the PureLink RNA mini isolation kit as recommended by the manufacturer. Then, 150 ng total RNA was labelled and hybridised to the Agilent Human SurePrint G3 Human GE 8 × 60 k microarray chip (Agilent Technologies, Santa Clara, CA). All microarray experiments were conducted at the Microarray Core Facility (Stem Cell Unit, King Saud University College of Medicine). Normalisation and data analyses were conducted using GeneSpring GX software (Agilent Technologies). Pathway analysis was conducted using the Single Experiment Pathway analysis feature in GeneSpring 12.0 (Agilent Technologies). A two-fold cut off with p < 0.05 was used.
Genespring 12
Manufactured by Agilent Technologies
Sourced in United States
GeneSpring 12.0 is a powerful software platform designed for the analysis and visualization of genomic data. It provides a comprehensive set of tools for data normalization, statistical analysis, and pathway-based interpretation of gene expression data. The software is optimized for handling large-scale datasets and offers a user-friendly interface for seamless integration with various data sources.
Automatically generated - may contain errors
Lab products found in correlation
Genespring gx software, by Agilent Technologies (6 mentions)
Feature extraction software 10, by Agilent Technologies (4 mentions)
Genechip 3 ivt express kit, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Agilent human sureprint g3 human ge 8 60 k microarray chip, by Agilent Technologies (3 mentions)
Agilent human sureprint g3 human ge 8 60 k v16 microarray chip, by Agilent Technologies (3 mentions)
Feature extraction 11, by Agilent Technologies (3 mentions)
Agilent microarray scanner, by Agilent Technologies (3 mentions)
Fluidics station 450, by Thermo Fisher Scientific (3 mentions)
Jmp 7, by SAS Institute (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Sureprint g3 human ge 8 60k microarray, by Agilent Technologies (2 mentions)
Purelink rna mini isolation kit, by Thermo Fisher Scientific (2 mentions)
Total rna purification kit, by Norgen Biotek (2 mentions)
Gene expression wash buffer kit, by Agilent Technologies (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Agilent 046065 mouse mirna microarray v19.0 8 60 k, by Agilent Technologies (2 mentions)
Genechip scanner 3000 7g, by Thermo Fisher Scientific (2 mentions)
Mirna complete labeling and hyb kit, by Agilent Technologies (2 mentions)
Prism software, by GraphPad (2 mentions)
79 protocols using genespring 12
1
TGF-β1 Induced Transcriptome Analysis
2
Transcriptome Profiling with GEDI Analysis
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The raw intensity data was exported to GeneSpring 12.0 (Agilent Technologies, Santa Clara, CA, USA) for quantile normalization. The normalized data containing 42544 probes were further analyzed using the R program. 139 positive control probes were removed. We then defined the coding (“NM_”, “XM_”) and non-coding (“lincRNA”, “NR_” and “XR_”) genes in the normalized data according to the definition of RefSeq accession format (http://www.ncbi.nlm.nih.gov/projects/RefSeq/key.html ). The landscape of whole transcriptome (lncRNAs + coding RNAs), lncRNAs only and coding RNAs only were analyzed with gene expression dynamic inspector (GEDI) [34 (link),35 (link)].
3
miRNA Profiling of Radiation-Treated HCT116 Cells
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MiRNA expression profiling by microarray analysis were conducted by a commercial service (Oebiotech). Briefly, small RNAs were isolated from HCT116 cell line after radiation, and then labeled with Cyanine-3-CTP. The fragmentation mixtures were hybridized to a Human miRNA Microarray 21.0 (8*60 K, Design ID: 070156). The feature extraction software 10.7.1.1 (Agilent) is used to analyze the scanned images. Raw data were normalized using Genespring 12.0 (Agilent). Student’s t test was used to identify the differential expression of miRNAs.
4
Microarray Analysis of Total RNA
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Total RNA was extracted as described above using Total RNA Purification Kit (Norgen-Biotek Corp.) according to the manufacturer's instructions. One hundred and fifty nanograms of total RNA was labeled and then hybridized to the Agilent Human SurePrint G3 Human GE 8 × 60 k v16 microarray chip (Agilent Technologies). All microarray experiments were conducted at the Microarray Core Facility (Stem Cell Unit, King Saud University College of Medicine). Normalization and data analyses were conducted using GeneSpring GX software (Agilent Technologies). Pathway analysis were conducted using the Single Experiment Pathway analysis feature in GeneSpring 12.0 (Agilent Technologies) as described before.33 (link), 34 (link) Twofold cutoff with P<0.02 was used.
5
Microarray Analysis of miRNA Expression
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Total RNA was analysed by microarray according to the manufacturer's protocol. Briefly, total RNA (100 ng) was labelled with the microRNA Complete Labelling and Hyb Kit (Agilent, Santa Clara, CA, USA), and then hybridized to the Agilent Human miRNA microarray V21.0 (8×60K) for 2549 human microRNAs. After hybridization, the microarray was washed using a Gene Expression Wash Buffer kit (Agilent). Hybridization signals were then scanned with the Agilent Microarray Scanner (Agilent) using Agilent scan control software Version A7.0 (Agilent). The data collection, background subtraction and within array normalization were performed by Agilent Feature Extraction Software (Agilent). Percentile normalization and principal component analysis were performed by Gene spring 12.0 (Agilent).
6
Transcriptome Analysis of Microarray Data
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Microarray data analysis was performed with the GeneSpring 12.0 platform (Agilent Technologies UK Ltd., South Queensferry, UK) to identify transcriptome differences between the control and the study groups. The oligonucleotide microarrays of Affymetrix HG-U133A enabled analysis of 22 283 mRNA transcripts. For further study, we selected only 98 transcripts from the Affymetrix NetAffx Analysis Center database (http://www.affymetrix.com/analysis/index.affx ). Normalized data were used to compile a list of 7 genes, the expression of which appeared to be up- or down-regulated by an arbitrary cutoff of at least 2-fold. Significant differential gene expression was identified by a 2.0-fold change at P<0.05. The results obtained through the oligonucleotide microarray technique were grouped with the hierarchical clustering method using the Euclidean distance measure.
The study was approved by the Bioethics Committee of the Medical University in Katowice, in accordance with the Declaration of Helsinki regarding medical research involving human subjects. The study and its purpose were explained to each participant or his or her legal guardian, who gave written informed consent.
The study was approved by the Bioethics Committee of the Medical University in Katowice, in accordance with the Declaration of Helsinki regarding medical research involving human subjects. The study and its purpose were explained to each participant or his or her legal guardian, who gave written informed consent.
7
RNA Microarray Analysis of PDX Tissues
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RNA microarray analysis of PDX tissue was performed as previously described [19 (link)]. Data are available at GEO accession number GSE41193. Total RNA samples were prepared and processed using Agilent's One-Color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labelling v6.0. An input of 100 ng of total RNA was used to generate cyanine-3-labeled cRNA. Samples were hybridized on Agilent SurePrint G3 Human GE 8×60K Microarray (Design ID, 028004). Arrays were scanned with the Agilent DNA Microarray Scanner at 3-μm scan resolution and data were processed with Agilent Feature Extraction 11.0.1.1. The processed signal was quantile normalized with Agilent GeneSpring 12.0.
8
Transcriptome Analysis of Biological Samples
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Ribonucleic acid from at least three biological replicates was independently collected as described above and its quality was assessed with NanoDrop (Thermo Scientific) and confirmed with RIN (RNA integrity number) at the University of Miami Center for Genome Technology (Miami, FL, USA). Amplification and processing for hybridization to GeneChip Human Gene ST 1.0 arrays (Affymetrix, Santa Clara, CA, USA) were performed at the University of Miami Center for Genome Technology (Miami, FL, USA) following standard protocols. Data were normalized with Genespring 12.0 (Agilent, Santa Clara, CA, USA) and filtered by intensity. Probes between the 20th and 99th percentile range in at least two of three samples per condition were included in the analysis. Statistical comparison between conditions was performed using unpaired Student's t-test with Benjamini-Hochberg correction for multiple testing. Final data analysis was conducted using Excel software (Microsoft Corporation, Redmond, WA, USA) and GeneGo (Thomson Reuters, Philadelphia, PA, USA).
9
miRNA Profiling of Bone Samples
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Small RNAs were isolated from the total RNA of HO and normal bone samples (five patients in each group). miRNAs were profiled using the Agilent Human miRNA Microarray V19.0 (Agilent, Santa Clara, CA, USA). The scanned images were analyzed with the Feature Extraction software 10.7.1.1 (Agilent) using default parameters to obtain background-subtracted and spatially detrended processed signal intensities as the raw data. The raw data were normalized with a quantile algorithm using Genespring 12.0 (Agilent). Probes for which at least 100% of the samples in any 1 of 2 conditions had flags in ‘Detected' were maintained.
10
Bovine Microarray and Upstream Regulator Analysis
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Microarray and upstream regulator gene analysis were performed following a previous study
[12 (link)]. Briefly, a customized bovine
oligonucleotide microarray (Agilent Technologies) was used to detect differentially
expressed genes (DEGs) in the RE using one-color microarray analysis as reported elsewhere
[13 (link)]. Fluorescently labeled (Cy3) complementary
RNA probes were hybridized, and the array was scanned using an Agilent Microarray Scanner
(Agilent Technologies). Feature Extraction ver. 9.1 software (Agilent Technologies) was
used to process the microarray images, align spots, and create raw numerical total spot
intensity data. The microarray data were imported into GeneSpring 12.0 (Agilent
Technologies), and normalization was performed (per-chip normalization). The Gene
Expression Omnibus accession numbers are as follows: platform, GPL22091; samples,
GSM2219049 to GSM2219055; and series, GSE83813 [12 (link)].
Lists of DEGs that corresponded to the raw estimated fold changes (FC; ≥2.0) were
uploaded into the Ingenuity Pathway Analysis (IPA) software (www.ingenuity.com; Ingenuity
Systems, Redwood City, CA, U.S.A.). The IPA knowledge base was used for DEG enrichment
analysis, and top upstream regulator analysis and statistical calculations were performed
to compare the two groups.
[12 (link)]. Briefly, a customized bovine
oligonucleotide microarray (Agilent Technologies) was used to detect differentially
expressed genes (DEGs) in the RE using one-color microarray analysis as reported elsewhere
[13 (link)]. Fluorescently labeled (Cy3) complementary
RNA probes were hybridized, and the array was scanned using an Agilent Microarray Scanner
(Agilent Technologies). Feature Extraction ver. 9.1 software (Agilent Technologies) was
used to process the microarray images, align spots, and create raw numerical total spot
intensity data. The microarray data were imported into GeneSpring 12.0 (Agilent
Technologies), and normalization was performed (per-chip normalization). The Gene
Expression Omnibus accession numbers are as follows: platform, GPL22091; samples,
GSM2219049 to GSM2219055; and series, GSE83813 [12 (link)].
Lists of DEGs that corresponded to the raw estimated fold changes (FC; ≥2.0) were
uploaded into the Ingenuity Pathway Analysis (IPA) software (www.ingenuity.com; Ingenuity
Systems, Redwood City, CA, U.S.A.). The IPA knowledge base was used for DEG enrichment
analysis, and top upstream regulator analysis and statistical calculations were performed
to compare the two groups.
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