To gain further insights into the specific genes affected by the miR-23b/27b/24-1 cluster, we performed a combination of in silico and genome-wide gene expression analyses. First, genes regulated by the miR-23b/27b/24-1 cluster were listed using the TargetScan database as described previously [15 (link), 38 (link), 39 (link)]. Next, to identify upregulated genes in PCa, we analyzed a publicly available gene expression data set in the Gene Expression Omnibus (GEO, accession number: GSE29079). Finally, we performed genome-wide gene expression analysis using PC3 cells transfected with miR-23b, miR-27b, or miR-24-1. A SurePrint G3 Human GE 60K Microarray (Agilent Technologies) was used for expression profiling of each miRNA transfectant in comparison with negative control miRNA transfectants. Finally, downregulated mRNAs that contained target sites for each miRNA (miR-23b/27b/24-1) were listed as putative target genes for these miRNAs.
Sureprint g3 human ge 60k microarray
Manufactured by Agilent Technologies
Sourced in Spain, United States
The SurePrint G3 Human GE 60K Microarray is a high-density gene expression microarray designed for comprehensive whole-genome profiling. It provides comprehensive coverage of human genes and transcripts, enabling researchers to analyze the expression of a large number of genes simultaneously.
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7 protocols using sureprint g3 human ge 60k microarray
1
Identifying miR-23b/27b/24-1 Target Genes
2
Identifying miR-145-3p Regulated Genes in mCRPC
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We performed a combination of in silico and genome-wide gene expression analyses. First, genes regulated by miR-145-3p were listed using the TargetScan database (release 7.0). Next, we performed genome-wide gene expression analysis using miR-145-3p-transfected PC3 cells. A SurePrint G3 Human GE 60 K Microarray (Agilent Technologies) was used for expression profiling of miRNA transfectants in comparison with mock PC3 cells(accession number: GSE77790, log2 ratio<−1.5). Next, we identified genes that were highly upregulated in mCRPC tissue compared with those in non-PCa and HSPC and highly upregulated in PCa compared with those in non-PCa tissues (accession number: GSE35988; fold change > 2 and q-value<0.05). Finally, to validate that these genes were upregulated in mCRPC clinical specimens, we used other data sets from cBioportal (http://www.cbioportal.org/ ) and GEO database (GSE 70770) (Ross-Adams et al, 2015 (link); Whitington et al, 2016 (link)). HSPC data were obtained from TCGA-PRAD (Cancer Genome Atlas Research N, 2015 (link)), and mCRPC data were obtained from SU2c/PCF (Robinson et al, 2015 (link)).
3
Gene Expression Analysis of Deadenylase Silencing
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Gene expression analysis of NCI-H520 and Hep2 cells upon deadenylase silencing was performed at NIMGenetics (Madrid, Spain) by using SurePrint G3 Human GE 60 K Microarray and Whole Human Genome Oligo Microarrays 44 k, respectively (Agilent Technologies, Inc., Santa Clara, CA). Initial analysis of the microarray data was performed with cut-off fold-change value ± 2. To exclude any possible transcripts affected by the transfection procedure itself, we used non-Τarget shRNA (Sigma), pLKO.1 empty vector (Sigma) and wild type cells as controls of expression. The microarray data have been deposited in the Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo , accession numbers GSE67536 and GSE67598). The upregulated genes arising from the deadenylases silencing were used to predict significantly enriched gene ontologies (GO). GO analysis was performed using GeneMANIA (www.genemania.org ) on February 1st 2015 [28 (link)].
4
Comprehensive Gene Expression Analysis
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Comprehensive gene expression analysis was performed at NIMGenetics (Madrid, Spain). Samples of NCI-H520 and HEp-2 cells upon deadenylase silencing were hybridized to microarrays SurePrint G3 Human GE 60 K Microarray, and Whole Human Genome Agilent 4 × 44 K oligo Microarray (Agilent Technologies, Inc., Santa Clara, CA, USA). To exclude any possible transcripts affected by the transfection procedure itself, we used non-target shRNA (Sigma), pLKO.1 empty vector (Sigma), and wild-type cells as controls of expression. Raw microarray data files are available in the GEO database with accession numbers GSE67536 and GSE67598 [50 (link),51 (link)].
5
Transcriptional Regulation by miR-451a
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The TargetScan database (Release 6.2; http://www.targetscan.org/ ) was used for in silico identification of candidate target genes that contained miR-451a targets sites in their 3′-untranslated (3′-UTR) region. To identify miR-451a-regulated genes, we used genome-wide gene expression analysis of FaDu cells transfected with miR-451a. SurePrint G3 Human GE 60K Microarray (Agilent Technologies) was used for expression profiling of miR-451a transfectants in comparison with negative control miRNA transfectants.
6
miR-452 Regulatory Gene Profiling
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We performed a combination of in silico and genome-wide gene expression analyses. First, genes regulated by miR-452 were listed using the TargetScan database. Next, to identify upregulated genes in PCa, we analysed a publicly available gene expression data set in GEO (accession number: GSE29079). Finally, we carried out genome-wide gene expression analysis using miR-452 transfectants of PC3 and DU145 cells. A SurePrint G3 Human GE 60K Microarray (Agilent Technologies) was used for expression profiling of miRNA transfectants in comparison with negative control miRNA transfectants. Finally, downregulated mRNAs containing miR-452 target sites were listed as putative target genes.
7
Identification of miRNA-221/222 Targets
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We performed a combination of in silico and genome-wide gene expression analyses. First, genes regulated by the miR-221/222 cluster were listed using the TargetScan database, as described previously (Kinoshita et al, 2013 (link); Nohata et al, 2013 (link); Goto et al, 2014a (link)). Next, to identify upregulated genes in PCa, we analysed a publicly available gene expression data set in GEO (accession number: GSE29079). Finally, we performed genome-wide gene expression analysis using miR-221- and miR-222-transfected PC3 and DU145 cells. A SurePrint G3 Human GE 60K Microarray (Agilent Technologies) was used for expression profiling of each miRNA transfectant in comparison with negative control miRNA transfectants. Finally, downregulated mRNAs containing miR-221/222 target sites were listed as putative target genes of these miRNAs.
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