The whole study design, including RNA extraction, microarray assay, and bioinformation steps were described before [11 (link),12 (link)]. In brief, total RNA was prepared with the miRNeasy Mini Kit (Qiagen, 40724 Hilden, Germany). For microarray analysis, we used the Agilent Array platform employing the manufacturer’s standard protocols for sample preparation and microarray hybridization. Gene expression analysis was performed with the Whole Human Gene Expression Microarray (4 × 44K; GPL4133), arrays were scanted with the Agilent G2505B Microarray Scanner and feature extraction was performed with Feature Extraction software version 9.5 (all Agilent Technologies, 76337 Waldbronn, Germany). Data files from mRNA microarrays were analyzed by GeneSpring GX 7.3.1 according to the manufacturer’s protocol (Agilent Technologies, 76337 Waldbronn, Germany). The first normalization step consisted of background elimination while, in a second step, the 50th percentile of each spot was normalized. Normalizations to a healthy oral mucosa pool was performed in the last step with the expression factor for the healthy oral mucosa pool set to 1; the fold change to control is presented in the tables. Primary statistical analysis was performed with GeneSpring GX 7.3.1 software (Agilent Technologies, 76337 Waldbronn, Germany).
Genespringgx 7
Manufactured by Agilent Technologies
Sourced in United States, Germany
GeneSpringGX 7.3 is a bioinformatics software tool designed for the analysis and visualization of genomic data. It provides a comprehensive suite of statistical and visualization tools for processing and interpreting complex gene expression data.
Automatically generated - may contain errors
Lab products found in correlation
Feature extraction software, by Agilent Technologies (21 mentions)
Dna microarray scanner, by Agilent Technologies (17 mentions)
Agilent dna microarray scanner, by Agilent Technologies (13 mentions)
Trizol reagent, by Thermo Fisher Scientific (10 mentions)
Rneasy mini kit, by Qiagen (8 mentions)
Agilent feature extraction software 10, by Agilent Technologies (7 mentions)
Agilent feature extraction software 9, by Agilent Technologies (5 mentions)
Mouse genome 430 2.0 array, by Thermo Fisher Scientific (4 mentions)
Rneasy kit, by Qiagen (3 mentions)
Low input quick amp labeling kit, by Agilent Technologies (3 mentions)
Rnaeasy column, by Qiagen (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Low rna input linear amplification kit, by Agilent Technologies (3 mentions)
Agilent g2505b microarray scanner, by Agilent Technologies (2 mentions)
Feature extraction software version 9, by Agilent Technologies (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
Hybridization solution, by Agilent Technologies (2 mentions)
Gasket 8 plex slide, by Agilent Technologies (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Recoverall total nucleic acid isolation kit, by Thermo Fisher Scientific (2 mentions)
98 protocols using genespringgx 7
1
Microarray Analysis of Oral Mucosa
2
Gene Expression Profiling of Cell Lines
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IHOK‐KGM, IHOK‐EF, and IHOK‐EFKGM were cultured to 70% confluency. Total RNA was isolated using RNeasy kit (Qiagen) according to the manufacturer's instructions. Each total RNA sample (200 ng) was labeled and amplified using Low Input Quick Amp labeling kit (Agilent technologies, CA). The Cy3‐labeled aRNAs were resuspended in 50 µl of hybridization solution (Agilent technologies). After labeled aRNAs were placed on Agilent Sure Print G3 Human GE 8 × 60K array (Agilent technologies) and covered by a Gasket 8‐plex slide (Agilent technologies), the arrays were analyzed using an Agilent scanner with associated software. Gene expression levels were calculated with Feature Extraction v10.7.3.1 (Agilent technologies). Relative signal intensity for each gene was generated using the Robust Multi‐Array Average algorithm. The data were processed based on the median polish normalization method using Gene Spring GX 7.3.1 (Agilent technologies). The normalized and log‐transformed intensity values were then analyzed using Gene Spring GX 7.3.1 (Agilent technologies).
3
Microarray Data Analysis Protocol
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Hybridization images were analysed with an Agilent DNA microarray Scanner (Agilent Technologies) and the data were quantified using Agilent Feature Extraction software 10.7 (Agilent Technologies). The average fluorescence intensity of each spot was calculated, and local background was subtracted. All data were normalised and fold-changes in gene expression were determined using GeneSpringGX 7.3.1 (Agilent Technologies). Normalization for Agilent one-color method was performed for data transformation: Set measurements less than 5.0 to 5.0 and Per Chip: Normalise to 50th percentage. The averages of normalised ratios were calculated by dividing the average of control normalised signal intensity by the average of test normalised signal intensity. Functional annotation of the genes was performed according to Gene Ontology Consortium (https://www.geneontology.org/index.shtml ) using GeneSpringGX 7.3.1 (Agilent Technologies).
4
Microarray Analysis of Arabidopsis Mutant
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Three-week-old WT and ospil1 plants grown under LD conditions were used for microarray analysis. Total RNA was extracted from the first leaves of WT and ospil1 plants using an MG Total RNA Extraction kit according to the manufacturer’s protocol (Macrogen, Korea). Total RNA quality was checked using a 2100 Bioanalyzer (Agilent Technologies). All microarray experiments, including data analysis, were performed according to the manufacturer’s manual (http://www.genomics.agilent.com/literature.jsp?crumbAction=push&tabId=AG-PR-1001&contentType=User+Manual ). The arrays were air-dried and scanned using a high-resolution array scanner (Agilent) with the appropriate settings for two-color gene expression arrays. GeneSpring GX 7.3 (Agilent) was used to calculate the intensity ratio and fold-changes, and quantified with the Feature Extraction Software (Agilent). For evaluating the statistical significance and obtaining the P-value, one-sample t-tests were performed using GeneSpring GX 7.3 (Agilent). Microarray analysis was performed with two experimental replicates with two different biological replicates of WT and ospil1 samples. Information about phytohormone- and photosynthesis-associated genes was obtained from the Oryzabase (www.shigen.nig.ac.jp/rice/oryzabase ).
5
Microarray Analysis of Rat Transcriptome
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Total RNA was isolated using Trizol reagent according to the manufacturer’s instructions (Invitrogen, Waltham, MA, USA). For microarray analysis, RNA was hybridized to Rat Oligo Microarray (44K) chips (Agilent Technology, Palo Alto, CA, USA). Next, the hybridized images were scanned using a DNA microarray scanner (Agilent Technology) and quantified with the Feature Extraction software (Agilent Technology). All data normalization and selection of fold-changed genes were performed using the GeneSpringGX 7.3 software (Agilent Technology). All genes meeting the cut-off criteria were further analyzed by hierarchical clustering and K-means clustering using the GENOWIZ Data Mining Tool (Ocimum Biosolutions, India). For functional analysis, genes were analyzed using DAVID (http://david.abcc.ncifcrf.gov/ ), Medline (http://www.ncbi.nlm.nih.gov/ ) and the KEGG (http://www.genome.jp/kegg/ ) database.
6
Gastric Cancer miRNA Profiling Protocol
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After manual dissection under microscopic guidance avoiding the contamination of inflammatory cells and stromal cells, Hematoxylin & eosin-stained sections, 50 mm in thickness from cancerous and noncancerous regions of intestinal type of gastric cancer formalin-fixed paraffin-embedded (FFPE) samples, were reviewed by one pathologist. Each section was incubated in xylene and total RNA was extracted using a RecoverAll™ Total Nucleic Acid Isolation Kit (Life Technologies, Carlsbad, CA, USA). Each 400 ng RNA was dephosphorylated with 15 units of calf intestine alkaline phosphatase, followed by RNA denaturation with 40% dimethylsulfoxide. Dephosphorylated RNA was ligated with pCp-Cy3 mononucleotide and resuspended in Gene Expression Blocking Reagent and Hi-RPM Hybridization buffer. The denatured and labeled samples were pipetted onto assembled Agilent Human miRNA microarray Release 16.0 platform and hybridized at 55°C for 20 hours at 20 rpm. The hybridization images were analyzed using a DNA microarray scanner (Agilent Technologies, Palo Alto, CA, USA). The average fluorescence intensity for each spot was calculated and local background was subtracted. Data visualization and analysis were performed with GeneSpring GX 7.3 software (Agilent Technologies). Signal cutoff measurements were P < 0.01.
7
Liver Transcriptome Analysis in HFD-Fed Mice
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Liver samples were collected from A/J male mice and A/J-12SM male mice fed the HFD for 7 weeks. Total RNA was isolated from the liver using TRI reagent (Molecular Research Center Inc.) and cleaned using an RNeasy Mini kit (Qiagen). We extracted the total RNA from nine mice per strain. Extracted total RNA quality was assessed with an Agilent Bioanalyzer (Agilent Technologies). cDNA synthesis and cRNA labeling reactions were performed with a One-Cycle cDNA Synthesis Kit and IVT Labeling Kit (Affymetrix). In order to minimize the experimental variation, we used three arrays for each strain and obtained data from three replicates. Each biotinylated cRNA was prepared from the pooled RNA samples of three mice. The biotinylated cRNAs were hybridized with a probe array (Mouse Genome 430 2.0 Array) by using a Hybridization, Wash and Stain kit (Affymetrix). Scanning was performed using a Scanner 3000 with GeneChip Operating Software (GCOS) (Affymetrix). Raw data were normalized with the MAS5.0 algorithm by using Expression Console Software (Affymetrix). The microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) (GSE67340). Data were analyzed by using GeneSpring GX7.3 software (Agilent Technologies).
8
Comparative Hypomethylation Analysis
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>I1.5I fold demethylated (hypomethylated) probes were independently analyzed in control (mock treated) vs. 5-aza-dC treated cells, control vs. 2 Gy irradiated cells or control vs. 5 Gy irradiated cells. The selected probes were 4,675, 89 and 71 probes, respectively. Commonly hypoemthylaed probes were collected between control vs. 5-aza-dC treated cells and control vs. 2 Gy irradiated cells, or between and control vs. 5 Gy irradiated cells via Venn diagram analysis, and finally common hypomethylated probes (25 and 21 probes, respectively) were analyzed. Hierarchical clustering was performed with Agilent GeneSpring GX 7.3 software using common hypomethylated probes. Pearson Correlation was used for similarity measurement.
9
Adipocyte transcriptome profiling
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Total RNA from fully differentiated C3H10T1/2 adipocytes treated with 20μM of butein, sulfurein or resveratrol for 6 hours were prepared using TRIzol and further purified using RNAeasy columns (QIAGEN). cDNA preparation and hybridization to Affymetrix Mouse Genome Arrays (430 version 2.0) were performed by Genochek. The data were analyzed using GeneSpring GX 7.3 software (Agilent Technologies). Microarray data sets were deposited at Gene Expression Omnibus (accession number GSE76672).
10
Quantitative Real-Time PCR and Microarray Analysis
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Total RNA was isolated from the cells or tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and an RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany). Total RNAs were used to synthesize the cDNA using a ReverTra Ace®qPCR RT Master Mix (TOYOBO, Osaka, Japan) with random primers. The cDNA was used to amplify selected genes with THUNDERBRID® SYBR® qPCR Mix (TOYOBO) and primers using the Applied Biosystems QuantStudio 3 Real-Time PCR (Applied Biosystems, Foster city, CA, USA). Expression levels and dene specific primer sets were described previously [28 (link),29 (link),30 (link)].
For the microarray analysis, total RNAs from C3H10T1/2 adipocytes treated with 10 μM of sesamol or tBHQ for 24 h were isolated and cleaned using an RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). To assess the reproducibility of the RNA amplification, three samples each of DMSO, tBHQ, and sesamol RNA were amplified independently. The cDNA preparation and hybridization to Affymetrix Mouse Genome Arrays of 430 version 2.0 were performed by Macrogen (Seoul, Korea). Data were analyzed using the GeneSpring GX 7.3 software (Agilent Technologies, Santa Clara, CA, USA).
For the microarray analysis, total RNAs from C3H10T1/2 adipocytes treated with 10 μM of sesamol or tBHQ for 24 h were isolated and cleaned using an RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). To assess the reproducibility of the RNA amplification, three samples each of DMSO, tBHQ, and sesamol RNA were amplified independently. The cDNA preparation and hybridization to Affymetrix Mouse Genome Arrays of 430 version 2.0 were performed by Macrogen (Seoul, Korea). Data were analyzed using the GeneSpring GX 7.3 software (Agilent Technologies, Santa Clara, CA, USA).
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