In order to identify the miR(s) specific to the esophageal mucosa of achalasia, total RNA was extracted from the biopsy mucosal tissues of 8 representative cases of achalasia and from those of 4 healthy volunteer controls. Following DNase treatment, the isolated RNA samples were subjected to comprehensive analysis of miR expression patterns using microarray-based technology. These analyses were performed by Hokkaido System Science Co., Ltd. (Sapporo, Japan) using the SurePrint G3 Human 8×60 K microarray version 2.0 (Agilent Technologies) and 50 ng aliquots of each total RNA sample. The scan was performed using the Agilent Technologies Microarray Scanner (Agilent Technologies) at 3 µm resolution, and each spot was digitized using Agilent Feature Extraction version 10.7.3.1 software. To identify the miRs that were differentially expressed in esophageal mucosa, data were imported to GeneSpring GX version 10.7.3.1 (Agilent Technologies) and analyzed; a feature is considered detected if the signal is three-fold greater than the error. The differences in miR expression were considered as statistically significant if the fold change in expression values was >2.0 and P<0.05 using a Student's t-test.
Feature extraction version 10
Manufactured by Agilent Technologies
Feature Extraction version 10.7.3.1 is a software tool developed by Agilent Technologies for the analysis of microarray data. It provides a comprehensive set of algorithms and tools for extracting and processing feature information from microarray images.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (5 mentions)
Low input quick amp wt labeling kit, by Agilent Technologies (4 mentions)
Microarray scanner, by Agilent Technologies (3 mentions)
Hybridization oven, by Agilent Technologies (3 mentions)
G2565ba scanner, by Agilent Technologies (2 mentions)
Suretag dna labeling kit, by Agilent Technologies (2 mentions)
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Low input quick amp kit, by Agilent Technologies (2 mentions)
Bioanalyzer 2000, by Agilent Technologies (2 mentions)
Sureprint g3 mouse gene expression 8x60k microarray, by Agilent Technologies (2 mentions)
Genespring gx, by Agilent Technologies (1 mentions)
Recoverall total nucleic acid isolation kit, by Thermo Fisher Scientific (1 mentions)
Agilent mirna labeling kit, by Agilent Technologies (1 mentions)
Genespring gx software version 11, by Agilent Technologies (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Agilent mirna microarrays, by Agilent Technologies (1 mentions)
Mirna labeling reagent and hybridization kit, by Agilent Technologies (1 mentions)
Genespring gx 12, by Agilent Technologies (1 mentions)
Sureprint g3 human ge 8 60 k, by Agilent Technologies (1 mentions)
23 protocols using feature extraction version 10
1
Profiling Esophageal Mucosa miRNAs in Achalasia
2
Transcriptomic Analysis of Spinal Cord Tissue
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The removed spinal cords were immediately dissected and fresh-frozen in liquid nitrogen. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany), following the manufacturer's protocol.
Subsequently, total RNA (80 ng) was amplified at 40°C for 2 hours using the Agilent Low RNA Input Quick Amplification Kit (Agilent Technologies, Santa Clara, Calif), following the manufacturer's protocol. Complementary RNA was labeled at 40°C for 2 hours with cyanine 3. The sample was incubated with a microarray slide (020908; Agilent Technologies) for 17 hours. Microarray signals were scanned in a microarray scanner (Agilent Technologies). The images were processed using Feature Extraction version 10.7.3.1 software (Agilent Technologies), which provided normalized cyanine 3 channel intensity values for each spot on an array. Data were normalized at the 75th percentile using GeneSpring version 14.5 (Agilent Technologies). Expression data were analyzed using the Functional Annotation Clustering tool in DAVID version 6.8 (https://david.ncifcrf.gov/ ), an online bioinformatics resource that provides functional and pathway enrichment analysis.24 (link) A P value <.05 was considered to indicate statistical significance on the basis of Bonferroni-adjusted P values. A gene count >2 served as the cutoff criterion, and the classification stringency was set to medium.
Subsequently, total RNA (80 ng) was amplified at 40°C for 2 hours using the Agilent Low RNA Input Quick Amplification Kit (Agilent Technologies, Santa Clara, Calif), following the manufacturer's protocol. Complementary RNA was labeled at 40°C for 2 hours with cyanine 3. The sample was incubated with a microarray slide (020908; Agilent Technologies) for 17 hours. Microarray signals were scanned in a microarray scanner (Agilent Technologies). The images were processed using Feature Extraction version 10.7.3.1 software (Agilent Technologies), which provided normalized cyanine 3 channel intensity values for each spot on an array. Data were normalized at the 75th percentile using GeneSpring version 14.5 (Agilent Technologies). Expression data were analyzed using the Functional Annotation Clustering tool in DAVID version 6.8 (
3
Profiling of miRNA Expression in Lung Metastasis
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miRNA expression profiling was conducted using the Human miRNA V19.0 Microarray (Platform GPL19730, G4872A; Agilent Technologies, Santa Clara, CA, USA), which consists of probes for 2006 human miRNAs based on Sanger miRBase release 19.0. Total RNA was extracted and purified from FFPE tissue using RecoverAllTM Total Nucleic Acid Isolation Kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. Microarray image information was converted to spot intensity values using Feature Extraction version 10.7 software (Agilent Technologies). Raw data were normalized by quantile algorithm using GeneSpring software version 12.6 (Agilent Technologies). Logarithmic transformation, using log base 2, was then analyzed. A paired-sample t test was used to identify miRNAs with significantly altered expression (fold change >1.5, P < 0.05). All microarray data were deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) [GEO:GSE80038]. Venny 2.1.0 (http://bioinfogp.cnb.csic.es/tools/venny/ ) was used to filter the specific lung metastasis-associated miRNAs for further analysis.
4
miRNA Expression Profiling in nHHDPCs
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In order to analyze the miRNA expression profile, nHHDPCs (2×106) were seeded into 60-mm culture dishes and treated with 400 μM PPD. Following 24 h of treatment, total RNA was purified using TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. Total RNA was dephosphorylated and labeled with pCp-Cy3 using an Agilent miRNA Labeling kit (Agilent Technologies, Inc., Santa Clara, CA, USA). Labeled RNAs were hybridized using a SurePrint G3 Human v16 miRNA 8×60K microarray (Agilent Technologies, Inc.) at 65°C for 20 h. The miRNA expression profile was digitized using Feature Extraction version 10.7 software (Agilent Technologies). Fold changes in miRNA expression levels were determined using GeneSpring GX software, version 11.5 (Agilent Technologies).
5
Fluorescent Labeling and Microarray Analysis of miRNA
6
miRNA Microarray Profiling of Plasma Samples
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100 ng of total RNA from each plasma sample was labeled and hybridized on human Agilent miRNA microarrays (8*60 K, Design ID: 046064) using the miRNA Labeling Reagent and Hybridization Kit (Agilent Technologies, USA) following the manufacturer’s protocol. Briefly, total RNA was dephosphorylated, denatured, and then labeled with Cy3-CTP. After purification, the labeled RNAs were hybridized onto the microarray. The hybridized array was then washed and scanned using high dynamic range settings according to Agilent specifications and data was extracted from the scanned image using Feature Extraction version 10.2 (Agilent Technologies). The miRNA array data were analyzed for data summarization, normalization, and quality control by GeneSpring GX 12.1 Software (Agilent Technologies). To select the differentially expressed genes, we used threshold values of 2-fold change (FC) and a paired t-test P value of 0.05.
7
Genome-wide Copy Number Profiling
8
RNA Extraction and Expression Analysis
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RNA was extracted and analyzed as previously described (Bjaanaes et al., 2016 ). In short, RNA isolation was done using standard TRIZOL methods and quantity and quality was controlled using the NanoDrop ND‐1000 spectrometer and the 2100 Bioanalyzer. Gene expression was assessed using gene expression microarrays from Agilent technologies (SurePrint G3 human GE 8 × 60 K and SurePrint G3 human GE v3 8 × 60 K for the AD and SCC respectively). The raw data were processed with the agilent's feature extraction Software with default parameters (Agilent feature extraction version 10.7.3.1). Probes were collapsed by median, samples were quantile normalized, and the data were log2 transformed. Since the samples from AD and SCC were analyzed using different versions of the platform, they were normalized separately.
9
Transcriptomic Analysis of MCF7 Cell Lines
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Total RNAs (50 ng) from MCF7-luc derivatives (MCF7-luc anti-NC, MCF7-luc anti-miR-27b, MCF7-luc miR-27b o.e. and MCF7-ENPP1-MF cells) were labelled and amplified using the Low Input Quick Amp labelling kit (Agilent Technologies). The Cy3-labelled RNAs were resuspended in 40 μl of hybridization solution (Agilent Technologies), applied to a SurePrint G3 Human GE 8 × 60 K array (Agilent Technologies) and covered with a Gasket 8-plex slide (Agilent Technologies). The slides were hybridized for 17 h at 65 °C, washed with Gene Expression Wash Buffer 1 (Agilent Technologies) for 1 min at room temperature, washed with Gene Expression Wash Buffer 2 (Agilent Technologies) for 1 min at 37 °C and then air-dried. The arrays from three independent experiments were analysed using microarray scanner (Agilent Technologies). Gene expression levels were calculated using Feature Extraction version 10.7.3.1 (Agilent Technologies). The normalized and log-transformed intensity values were then analysed using GeneSpring GX 7.3.1 (Agilent Technologies).
10
Robust Microarray Gene Expression Profiling
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Within each sampling tier (as defined above), RNA samples were randomized prior to labeling, then randomized again prior to hybridization, to avoid any potential batch effects associated with either the labeling or hybridization procedures. Total RNA (50ng) was amplified and Cy 3 labeled using Agilent’s Low Input Quick Amp Labeling Kit. The Cy3 labeled cRNA was quantified using a Nanodrop ND-1000 spectrophotometer. A total of 1.65 μg of Cy3 labeled cRNA was hybridized to the microarray. The One-Color Microarray-Based Gene Expression Analysis Protocol (Version 6.5, May 2010) was followed for both the labeling and the hybridization. Hybridization was carried out at 65°C in an Agilent hybridization oven rotating at 10 rpm for 17 hours. Slides were scanned with an Agilent G2505B scanner equipped with Agilent Scan Control software, using the Extended Dynamic Range Scan Mode, 5μM scan, XDR PMT Hi 100% Lo 10%. Images were extracted using Agilent Feature Extraction version 10.7.3.1.
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