miRNA expression was investigated using an Agilent Human miRNA Microarray #G4870C and Sanger miRBase v.21 (Agilent Technologies). Microarray data are available at the ArrayExpress database (https://www.ebi.ac.uk/arrayexpress) with the accession number E-MTAB-7585. RNA labeling and hybridization were performed in accordance with the manufacturer’s instructions. The microarray results were analyzed with GeneSpring software v.14.9 (Agilent Technologies). Quantile normalization and log2 transformation were applied. Differentially expressed miRNAs were selected that had a 1.5-fold difference between CD99pos- and CD99neg EXOs and an adjusted p value < 0.20 in a moderated t-test with Benjamini and Hoechberg correction. Hierarchical clustering was performed using Pearson’s centered correlation as a measure of similarity.
Genespring software v 14
Manufactured by Agilent Technologies
Sourced in United States
GeneSpring software v.14.9.1 is a bioinformatics software tool for the analysis and visualization of genomic data. It provides a comprehensive suite of data analysis capabilities to support researchers in the interpretation of complex biological data.
Automatically generated - may contain errors
Lab products found in correlation
Genechip human transcriptome array 2, by Thermo Fisher Scientific (1 mentions)
Metacore, by Thomson Reuters (1 mentions)
Genechip human genome u133 plus 2.0 array, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Spss v16, by IBM (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Feature extraction software version 10, by Agilent Technologies (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
5 protocols using genespring software v 14
1
Differential miRNA Profiling of CD99+ Exosomes
2
Transcriptome Analysis of TC-71 Cells
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Gene expression patterns were assessed using a GeneChip® Human Transcriptome Array 2.0 (Affymetrix, Santa Clara, CA, USA) starting from 100 ng of total RNA. Microarray data are available at the GEO database (https://www.ncbi.nlm.nih.gov/geo) with the accession number # GSE128681. The microarray target sample processing, target hybridization, washing, staining, and scanning steps were completed according to the manufacturer's instructions (Affymetrix). Raw data were analyzed with GeneSpring software v.14.9 (Agilent Technologies, Santa Clara, CA, USA). Specifically, .CEL files were imported and processed using the ExonRMA16 summarization algorithm. Quantile normalization was applied without baseline transformation. Differentially expressed genes were defined as having a ≥ 1.5-fold expression difference between groups and an adjusted p-value of less than 0.05 in a moderated t-test with Benjamini and Hoechberg correction for false positive reduction. Hierarchical clustering was performed for the TC-71 samples with the GeneSpring clustering tool using Pearson’s centered correlation as a measure of similarity. Pathway and network analyses of the differentially expressed genes were performed using the web-based software MetaCore (GeneGo, Thomson Reuters).
3
Transcriptome Analysis of TRS Cells
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Total RNA from TRS cells was extracted by the RNeasy kit (Qiagen, Hilden, Germany). Microarray analysis was performed using the GeneChip Human Genome U133 Plus 2.0 Array (Thermo Fisher Scientific) according to the manufacturer’s instructions. GeneSpring software v.14.9.1 (Agilent, Santa Clara, CA) was used for quantile normalization and data processing. Gene ontology (GO) analysis was performed using Metascape. Gene set enrichment analysis (GSEA) was performed using GSEA v4.1.0 software. PI3K signaling and apoptosis signatures were downloaded from MSigDB: Hallmark mTORC1 signaling, Hallmark PI3K/Akt/mTOR signaling, Reactome Apoptosis and Reactome Regulation of apoptosis.
4
Differentially Expressed Gene Analysis
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The microarray data were statistically analysed using Welch ANOVA followed by Tukey’s HSD test with Benjamini-Hochberg multiple testing corrections for false discovery rate to identify differentially expressed (DE) genes having > 2-fold at P < 0.05 in pair-wise comparisons. All data processing and analysis were performed using the GeneSpring software v14.9.1 (Agilent, Santa Clara, USA). Differential expression for RNA-seq data for Y chromosome mapped reads were analysed using the software DESeq v1.31.0 (Bioconductor Package, Buffalo, NY, USA). Differentially expressed genes were subjected to the Wald test followed by a Benjamini-Hochberg correction. A cut-off value of > 2-fold change and P < 0.05 were used to report the final list of significantly differentially expressed genes. Inter-group differences in quantitative RT-PCR were calculated as described previously [20 (link)]. For statistical analysis of prevalence, the Fisher’s exact test was performed. For non-Gaussian data, the Mann-Whitney U-test was applied, using SPSS v16.0 (IBM Analytics, New York, US).
5
Transcriptional Profiling of Hypoxia-Induced Changes
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RNA concentration and integrity of the cells were checked using Nanodrop (Thermo-Fisher Scientific, MA, USA) and Bioanalyzer (Agilent, Santa Clara, CA, USA) respectively.
Expression microarray was done on two biological replicates each, of human FNSCs exposed to normoxia and hypoxia, on 4 x 44K human expression array slides (G2519F) (Agilent, Santa Clara, CA, USA), following manufacturer's protocol. Briefly, a total of 200 ng total RNA per sample (n=2) were subjected to cDNA conversion and linear amplification with fluorescent labeling for preparing Cy3 labeled cRNA. Complementary hybridization (17 hours at 65 °C) was followed by washing. Slides were scanned at 3 µm resolution, followed by feature extraction using feature extraction software version 10.7.1.1 (Agilent technologies, Santa Clara, USA). Analysis was done using Genespring Software v14.9.1 (Agilent technologies, Santa Clara, USA). Principal component analysis and Hierarchical clustering analysis were done to identify gene target distribution, that showed a consistent difference in expression between normoxia and hypoxia exposed cells. This was followed by differential gene expression analysis, which was done using unpaired t-test using Genespring software (GX v14.9.1). The list of differentially expressed genes were imported into gene onotolgy consortium and Metacore for gene ontology and pathway analysis.
Expression microarray was done on two biological replicates each, of human FNSCs exposed to normoxia and hypoxia, on 4 x 44K human expression array slides (G2519F) (Agilent, Santa Clara, CA, USA), following manufacturer's protocol. Briefly, a total of 200 ng total RNA per sample (n=2) were subjected to cDNA conversion and linear amplification with fluorescent labeling for preparing Cy3 labeled cRNA. Complementary hybridization (17 hours at 65 °C) was followed by washing. Slides were scanned at 3 µm resolution, followed by feature extraction using feature extraction software version 10.7.1.1 (Agilent technologies, Santa Clara, USA). Analysis was done using Genespring Software v14.9.1 (Agilent technologies, Santa Clara, USA). Principal component analysis and Hierarchical clustering analysis were done to identify gene target distribution, that showed a consistent difference in expression between normoxia and hypoxia exposed cells. This was followed by differential gene expression analysis, which was done using unpaired t-test using Genespring software (GX v14.9.1). The list of differentially expressed genes were imported into gene onotolgy consortium and Metacore for gene ontology and pathway analysis.
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