Total RNA was isolated using a miRNeasy Kit (Qiagen, Valencia, California, USA) according to the manufacturer’s instructions. Quality-confirmed total RNA samples were determined with the Quant-iT™ RNA Assay kit (Invitrogen, Carlsbad, California, USA). The samples were labeled with cyanine 3-pCp and subsequently hybridized to the Agilent mouse microRNA microarray release version 15 (1881 mouse miRNAs represented) using a microRNA Complete Labeling and Hyb Kit (Agilent Technologies) for 20 h. After washing, the slides were scanned with a G2565BA scanner, and the data were analyzed and monitored with Agilent Feature Extraction Software version 9.5.1 and GeneSpring GX software version 12.5 (Agilent Technologies).
Genespring gx software version 12
Manufactured by Agilent Technologies
Sourced in United States
GeneSpring GX software version 12.5 is a bioinformatics software tool designed for the analysis of gene expression data. It provides a comprehensive suite of tools for data normalization, statistical analysis, and visualization. The software is capable of handling a wide range of gene expression data formats and supports multiple analytical approaches.
Automatically generated - may contain errors
Lab products found in correlation
Microrna complete labeling and hyb kit, by Agilent Technologies (2 mentions)
Agilent feature extraction software version 9, by Agilent Technologies (2 mentions)
Mirneasy kit, by Qiagen (2 mentions)
Quant it rna assay kit, by Thermo Fisher Scientific (1 mentions)
Gene expression wash buffer, by Agilent Technologies (1 mentions)
Agilent mouse microrna microarray, by Agilent Technologies (1 mentions)
Agilent scanner g2565ba, by Agilent Technologies (1 mentions)
Ingenuity pathway analysis (ipa), by Qiagen (1 mentions)
G2505c dna microarray scanner, by Agilent Technologies (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Trizol solution, by Thermo Fisher Scientific (1 mentions)
Expression console version 1, by Thermo Fisher Scientific (1 mentions)
High pure rna isolation kit, by Roche (1 mentions)
Human genome u133 plus 2.0 genechip, by Thermo Fisher Scientific (1 mentions)
8 protocols using genespring gx software version 12
1
Microarray Analysis of Mouse miRNA
2
Mouse miRNA Expression Profiling
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Total RNA was isolated using a miRNeasy kit (Qiagen). After dephosphorylation and denaturation, the total RNA was labeled with cyanine 3-pCp and subsequently hybridized to an Agilent mouse microRNA microarray (release version 15) using the microRNA Complete Labeling and Hyb Kit (Agilent Technologies, Inc.). After hybridization for 20 h, the slides were washed using the Gene Expression Wash Buffer (Agilent Technologies, Inc.), scanned using an Agilent Scanner G2565BA, and processed and analyzed using Agilent Feature Extraction Software version 9.5.1. The raw data were analyzed using GeneSpring GX software version 12.5 (Agilent Technologies, Inc.).
3
Microarray Data Analysis of Cultured Islets
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Normalization and filtering of microarray data were performed using Gene Spring GX software Version 12.5 (Agilent Technologies, Santa Clara, CA, USA). Differentially expressed genes were defined as those levels that changed > 2-fold or ≤ 2-fold (log2) compared with the respective control. Ingenuity Pathway Analysis (Qiagen, Redwood City, CA, USA; qiagen.com/ingenuity ) was used to identify the biological processes that were differentially affected in MMC- or nontreated cultured islets. This tool provides information about a disease, molecular and cellular functions, and physiological and development function categories related to genes obtained from the microarray analysis. To determine the biological functions most closely associated with MMC-induced engraftment, we used an activation z-score of >2 or ≤2 and P value of <0.05. The activation z-score assesses the match between observed and predicted upregulation or downregulation patterns that indicate significant differential biological activity of MMC-treated islets compared with that of control islets (nontreated d0-islets). The P value, which was calculated using the Fisher exact test, indicates the statistical significance of the association of a biological function with a set of differentially expressed genes.
4
Profiling miRNA Expression in Cell Media
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CM from irradiated and control A549 cells 24 h after IR were collected for miRNA expression profile assay. Total RNA was extracted from 8 ml CM using a mirVana miRNA Isolation Kit (Ambion Inc., TX, USA) and analyzed with a miRNA microarray kit (Release 19.0, Agilent Technologies, CA, USA) described earlier [35 (link)]. There are 2006 human miRNAs on this microarray (Design ID: 046064). Briefly, 200 ng of total RNA was labelled and hybridized using the miRNA microarray kit. Hybridization signals were detected with a DNA microarray scanner G2505C (Agilent Technologies) and the scanned images were analyzed using the Agilent feature extraction software (v10.7.3.1). Normalization was performed using the Agilent GeneSpring GX software version 12.5.
5
RNA Isolation and Microarray Analysis of hMSC Synovium
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RNA was isolated from synovium 1, 3, and 7 days after injection of 1 Â 10 6 hMSCs (n ¼ 4, each) using TRIzol solution (Invitrogen Life Technologies, Carlsbad, CA, USA) and High Pure RNA Isolation Kit (Roche Applied Sciences, Indianapolis, IN, USA). A microarray analysis was performed using 500 ng of total RNA from each sample and GeneChip ® Rat Genome 230 2.0 probe arrays (Affymetrix, Santa Clara, CA, USA) and/or GeneChip ® Human U-133 plus 2.0 probe arrays. Data was analyzed with GeneSpring GX software version 12.5 (Agilent Technologies, Palo Alto, CA, USA). To normalize the variations in staining intensity among chips, the signal values for all genes on a given chip were divided by the median value for the expression of all genes on the chip. To eliminate genes containing only background signals, genes were selected only if the raw values of the signal were more than the lower limit of the confidence interval, and expression of the gene was judged to be 'present' using MAS5 algorithm in Expression Console Version1.1 (Affymetrix). The microarray data were deposited in the Gene Expression Omnibus (GEO accession no. GSE61617).
6
Ago2-RISC Immunoprecipitation and Microarray Analysis
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Immunoprecipitation (IP) of the Ago2-containing RISC was performed in four HL cell lines (L1236, L428, L540, and KM-H2), as described previously using 30 million cells as input. 28 Microarray analysis was performed as previously described. 29 Briefly, cRNA was synthesized from total (T) and IP fractions of four HL cell lines. This was followed by a cRNA amplification and labeling step with cyanine 3-CTP (Cy3) or cyanine 5-CTP (Cy5). Equal amounts of Cy3-or Cy5-labeled cRNA were mixed and hybridized on Human Whole Genome Oligo Microarray overnight (SurePrint G3 Custom GE 8 Â 60K; Agilent Technologies). Quantile normalization of signals was performed using GeneSpring GX software version 12.5 (Agilent). Probes with inconsistent intensities in Cy3 and Cy5 replicates and probes not detected in either IP or total fractions were filtered out. For the consistent probes expressed above the background, the average signals for Cy3 and Cy5 replicates were used to calculate IP/T ratio for each sample. The Ago2-RIPechromatin IP data were deposited in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih. gov/geo; accession number GSE92615).
7
Microarray Data Analysis Protocol
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GeneSpring GX software, version 12 (Agilent Technologies) was used for microarray data analysis. RMA normalization was used for mRNA data and quantile normalization for miRNA data. Statistically significant differentially expressed genes identified by moderated t-Test combined with the Benjamini and Hochberg correction for multiple testing and using filters based on P-value cut-off 0.05 and fold change cut-off +/-1.5.
8
Epigenetic Drug Effects on Cancer Cell Transcriptome
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Cancer cell lines (Additional file 5 : Table S4) were treated with epigenetic drugs and RNA expression was profiled on Affymetrix Human Genome U133 Plus 2.0 GeneChip® microarrays (Affymetrix, Santa Clara, CA) as described [56 (link)]. Normal tissue DNA and RNA were purchased from Amsbio (Abingdon, UK). Microarray data were analyzed by GeneSpring GX software, version 12 (Agilent Technologies, Santa Clara, CA) using RMA normalization. Statistically significant gene expression changes were identified by moderated t test combined with the Benjamini and Hochberg correction for multiple testing and by using filters based on p value cutoff of 0.05 and fold change cutoff of +/−1.5. The mRNA expression profiling data have been submitted to GEO (accession number: GSE58058).
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