Total RNA containing small RNA was extracted from stable transfectant HT-29 cells by using Trizol reagent (Invitrogen) and purified with mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). The Agilent 8 × 60 K mRNA microarray was constructed at CapitalBio Corporation (Beijing, China). The array data were analyzed for data summarization, normalization, and quality control by using the GeneSpring software V13 (Agilent). To select the differentially expressed genes, we used threshold values of ≥ 2 and ≤ −2-fold change, and Benjamini–Hochberg corrected p-value ≤ 0.05.
Genespring software v13
Manufactured by Agilent Technologies
Sourced in United States, China
GeneSpring software V13.0 is a data analysis platform designed for life science and bioinformatics applications. The software provides tools for the analysis and visualization of genomic, transcriptomic, and other biological data. GeneSpring V13.0 offers a comprehensive suite of data processing and statistical analysis functionalities to support researchers in their work.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (13 mentions)
2100 bioanalyzer, by Agilent Technologies (6 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (5 mentions)
Human circrna array v2, by CapitalBio (5 mentions)
Crna amplification and labeling kit, by CapitalBio (4 mentions)
Agilent human mrna array, by Agilent Technologies (3 mentions)
Agilent microarray scanner, by Agilent Technologies (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Agilent human lncrna mrna array v4, by Agilent Technologies (3 mentions)
Agilent feature extraction software version 10, by Agilent Technologies (2 mentions)
Nucleospin rna clean up kit, by Macherey-Nagel (2 mentions)
Feature extraction v10.7 software, by Agilent Technologies (2 mentions)
Agilent scanner g2505c, by Agilent Technologies (2 mentions)
Melatonin, by Merck Group (2 mentions)
Genechip mouse genome 430 2.0 array, by Agilent Technologies (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (2 mentions)
Agilent human circrna array v2, by Agilent Technologies (2 mentions)
G2565ca microarray scanner, by Agilent Technologies (2 mentions)
Rnaiso plus kit, by Takara Bio (2 mentions)
Feature extraction software, by Agilent Technologies (2 mentions)
66 protocols using genespring software v13
1
Differential Gene Expression Analysis
2
Transcriptome Analysis of DMSO and STM Treated HepG2 Cells
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After HepG2 cells were treated with DMSO or STM, total RNA from each sample was isolated using Trizol (Invitrogen) and purified using mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA) following the manufacturer's instructions. RNA integrity was determined by denatured agarose gel electrophoresis. DNA microarray was performed according to standard protocol.
CapitalBio cRNA Amplification and Labeling Kit (CapitalBio) was used to produce fluorescent dye labeled cDNA. The labeled cDNAs were then hybridized to Agilent human mRNA Array which was designed with eight identical arrays per slide (8 x 60K format). After hybridization at 42°C overnight, the arrays were washed with 2X SSC and 0.2% SDS at 42°C for 5 min, followed by washing with 0.2X SSC and 0.2% SDS at room temperature for 5 min. The array data were analyzed for data summarization, normalization and quality control by using the GeneSpring software V13 (Agilent).
CapitalBio cRNA Amplification and Labeling Kit (CapitalBio) was used to produce fluorescent dye labeled cDNA. The labeled cDNAs were then hybridized to Agilent human mRNA Array which was designed with eight identical arrays per slide (8 x 60K format). After hybridization at 42°C overnight, the arrays were washed with 2X SSC and 0.2% SDS at 42°C for 5 min, followed by washing with 0.2X SSC and 0.2% SDS at room temperature for 5 min. The array data were analyzed for data summarization, normalization and quality control by using the GeneSpring software V13 (Agilent).
3
Profiling miRNA Expression via Agilent Array
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Total RNA containing small RNAs was extracted from cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, United States) and purified with the mirVana miRNA Isolation Kit (Ambion, Austin, TX, United States) according to the manufacturer’s protocol. miRNA profiling was performed using an Agilent miRNA array, which was designed with eight identical arrays per slide, with each array containing 2549 human mature miRNA probes and 2164 Agilent control probes. Each miRNA was detected by probes and was repeated 30 times. Microarray experiments were conducted according to the manufacturer’s instructions. Briefly, total RNA (200 ng) was dephosphorylated and ligated to pCp-Cy3, and the labeled RNA was purified and hybridized to miRNA arrays. Images were scanned with an Agilent microarray scanner (Agilent, CA, United States) and gridded and analyzed using Agilent feature extraction software version 10.10. The miRNA array data were processed for data summarization, normalization, and quality control by using the GeneSpring software V13 (Agilent, CA, United States). To select the differentially expressed genes, we used threshold values of ≥2- and ≤−2-fold change and a Benjamini–Hochberg corrected p-value of 0.05. A clustering analysis was performed using Cluster 3.0 and visualized by Tree-View software.
4
Microarray Data Analysis Workflow
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The array data were analyzed for summarization, normalization, and quality control using GeneSpring software V13 (Agilent). To select differentially expressed genes, we used threshold values of ≥2‐fold and ≤−2‐fold change and a Benjamini‐Hochberg‐corrected P value of 0.05. The data were log2‐transformed and median‐centered by genes using the Adjust Data function of CLUSTER 3.0 software, then further analyzed with hierarchical clustering with average linkage. Finally, we performed tree visualization using Java Treeview (Stanford University School of Medicine, Stanford, CA).
5
Identifying LINC-PINT Regulated Genes
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To identify genes regulated by LINC-PINT, LINC-PINT-overexpressing HNE1 cells and control group cells were selected for microarray analysis. In Brief, total cellular RNA was extracted using the RNAiso Reagent (Takara, Japan), and qualified RNA was then purified by mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). After purification, cDNA was amplified using a poly dT-T7 promoter primer and covalently coupled with Cyanin3 (Cy3). Labeled cDNA samples were then subjected to SurePrint G3 Human Gene Expression 8x60K v2 Microarray Kit (Agilent). For downstream analysis, gene array data were analyzed and filtered using the GeneSpring software V13 (Agilent) according to the manufacturer’s instructions. Relative to control, signal intensity >onefold change and P < 0.05 were considered as differentially expressed genes, which were induced by LINC-PINT overexpression. For functional gene enrichment, GO analysis was performed and the P value was calculated by Bonferroni corrected Fisher’s exact tests.
6
Transcriptome Analysis of VP35 in HepG2 Cells
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The SurePrint G3 human gene expression 8x60k v2 microarray from Agilent was used for transcriptome analysis. Briefly, HepG2 cells were transfected with the plasmids Flag-VP35 or Flag. Forty-eight hours later, total RNA was extracted using the RNeasy mini kit (QIAGEN, USA) according to the manufacturer’s protocol. After first-strand cDNA synthesis with Poly-dT primer and second-strand cDNA synthesis, the cDNA was subjected to Gene Expression Microarray analysis following the manufacturer’s protocol. The chips were scanned with a GeneChip Scanner G2565CA (Agilent) and analyzed with Agilent Feature Extraction (v10.7) and GeneSpring software V13 (Agilent).
7
Transcriptome Analysis of hADSCs and CD146+ Cells
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Total RNA was isolated from hADSCs and freshly sorted CD146+ cells using Trizol reagent and purified with mirVana mRNA Isolation Kit (Ambion, Austin, TX, USA). The RNA integrity was determined using the RNA 6000 Nano Lab-on-a-Chip kit and the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Only RNA extracts with RNA integrity number values >6 were used for further analysis. Gene expression analyses were performed using the Agilent human mRNA Array. The array data were analyzed with the help of Capitalbiotech for data summarization, normalization, and quality control by using the GeneSpring software V13 (Agilent). To select the differentially expressed genes, we used threshold values of ≥2- and ≤0.5-fold change and a Benjamini-Hochberg-corrected P-value of 0.05. The data were log(2)-transformed and median-centered by genes using the Adjust Data function of CLUSTER 3.0 software. The data were further analyzed with hierarchical clustering with average linkage 38 (link). Tree visualization was performed by using Java Treeview (Stanford University School of Medicine, Stanford, CA, USA). Gene ontology (GO) pathway enrichment analysis was performed by WEGO (http://wego.genomics.org.cn/cgi-bin/wego/index.pl ).
8
Agilent miRNA Expression Profiling Protocol
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Agilent miRNA array was used for the miRNA profiling in accordance with the manufacturer's instructions. Each slide of the Agilent array has eight identical arrays (8 × 60 K format), and each array contains probes that interrogates 2549 human mature miRNAs from miRBase R21.0. Each miRNA was detected by probes repeat for 30 times. 2164 Agilent control probes are also included in the array. In a nutshell, the Agilent miRNA labeling reagent was used to label the miRNAs. The labeled RNA was isolated and hybridized to miRNA arrays after being dephosphorylated and ligated with pCp-Cy3 using 200 ng of total RNA. Agilent feature extraction software version 10.10 was used to analyze the images after they had been scanned using the Agilent microarray scanner (Agilent). The GeneSpring software V13 (Agilent) was used to analyze the miRNA array data for data summarization, standardization, and quality control (Agilent).
9
Differential Gene Expression Analysis
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The miRNA and mRNA array data were analyzed for data summarization, normalization, and quality control using GeneSpring software V13 (Agilent). The default 90th percentile normalization method was performed for data preprocessing. To select the differentially expressed genes, we used fold change threshold values of ≥2 and ≤−2 and a Benjamini–Hochberg corrected P = 0.05. The data were Log2-transformed and median centered by genes using the Adjust Data function in CLUSTER 3.0 software (Stanford University, Palo Alto, CA, USA) and were then further analyzed via hierarchical clustering with average linkage. To better understand the roles of the differentially expressed mRNAs, Gene Ontology (GO) categories derived from the GO database (www.geneontology.org) were determined, and pathway analysis was performed. Three families of GO terms were identified: biological process, cellular component, and molecular function.20 (link) Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/ ) analysis allowed us to determine the biological pathways that were significantly enriched with differentially expressed mRNAs.21 (link) Disease enrichment was analyzed by a web server, KOBAS 2.0 (http://kobas.cbi.pku.edu.cn/ ), which annotates an input set of genes with putative pathways and disease relationships based on mapping to genes with known annotations.22 (link)
10
Profiling of miRNA in NPC Radiosensitivity
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Total RNA samples were analyzed by CapitalBiotech (CapitalBio Corp.) for microarray experiments. The Agilent human miRNA Array V21.0 (8 × 60 K) was applied for detection of profiling of miRNA in the serum specimens of 12 NPC patients with different radiosensitivity. Totally, the microarray covered 2,549 miRNAs from the latest miRBase database. The slide was scanned by the Agilent Microarray Scanner (Cat# G2565CA, Agilent technologies, Santa Clara, CA, US) and the Feature Exaction software 10.7 (Agilent technologies, Santa Clara, CA, US)16 (link). The miRNA array data were analyzed for data summarization, normalization and quality control by using the GeneSpring software V13 (Agilent). The default 90th percentile normalization method were performed for date preprocessing. All steps were performed were according to the recommended protocol. To screen out the candidate miRNAs, threshold values of ≥2 and ≤−2 fold change and a Benjamini-Hochberg corrected p vlaue of 0.05 were used. The data was Log2 transformed and median centered by genes using the Adjust Data function of CLUSTER 3.0 software. Then we further analyzed the data with hierarchical clustering with average linkage. Finally, we performed tree visualization by using Multiple Experiment Viewer (MeV version 4_9_0).
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