To investigate the miRNA expression profile, microarrays were performed as previously described.14 (link) In brief, 100 ng of total RNA was used to construct miRNA libraries, which were then investigated with the Human microRNA Microarray Kit (version 3; Agilent Technologies, Santa Clara, CA, USA), in accordance with the manufacturer’s instructions. Microarray data were processed for standardization, quality evaluation, and statistical analysis. The 75% quartile method was used for normalization. Analysis of miRNAs that regulate VEGF expression was performed using Targetscan Human 7.2 (www.targetscan.org/vert_72/ ).
Human microrna microarray kit
Manufactured by Agilent Technologies
Sourced in United States
The Human microRNA Microarray Kit provides a comprehensive solution for the detection and profiling of human microRNA (miRNA) expression. The kit includes an array of oligonucleotide probes designed to capture and quantify a wide range of known human miRNAs. It enables researchers to analyze the expression levels of miRNAs in various biological samples, such as cells, tissues, or biofluids.
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Lab products found in correlation
Nanodrop nd 2000, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Total rna purification kit, by Norgen Biotek (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Human mirna microarray kit, by Agilent Technologies (1 mentions)
Genespring gx software, by Agilent Technologies (1 mentions)
G2505c dna microarray scanner, by Agilent Technologies (1 mentions)
C scanner, by Agilent Technologies (1 mentions)
Agilent feature extraction software, by Agilent Technologies (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Whole human genome oligo microarray, by Agilent Technologies (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
6 protocols using human microrna microarray kit
1
Profiling miRNA Expression via Microarray
2
Profiling miRNA in Esophageal Cancer
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Total RNA (100 ng) was labeled and hybridized following the Human microRNA Microarray Kit protocol and using the Human miRNA Microarray Kit (Release 16.0; Agilent Technologies). Hybridization signals were detected with a DNA microarray scanner G2505C (Agilent Technologies), and the scanned images were analyzed using the Agilent Function Extraction Software program (v10.7.3.1). Normalization was performed using the Agilent GeneSpring GX software program version 11.0.2 (per chip: Normalization of control genes; per gene: None). The Agilent Human miRNA Microarray (Design ID: 031181) contained 1205 human miRs in total with 144 human viral miRs without control probes. The miR expression profile of the serum samples from patients with ESCC was examined using a microarray analysis of three patients and four healthy individuals. miR-16 was used as an endogenous control.
3
Exosomal RNA Extraction and Microarray Analysis
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Total RNA Purification Kit (Norgen, CA) was used to extract exosomal RNA according to the manufacturer's protocols. Total RNA of cells was extracted using Trizol Reagent (Invitrogen, USA). The quantity and quality of RNA was assessed by NanoDrop® ND-2000 (Thermo Scientific) and Agilent 2100 Bioanalyzer (Agilent Technologies). The Human microRNA Microarray Kit (Agilent Technologies) was used for labeling and hybridization according to the manufacturer's protocol. In brief, 100 ng total RNA was labeled with Cyanine3 (Cy3), re-suspended in hybridization buffer and hybridized to the array platform overnight (20 h) at 55°C in a rotating Agilent hybridization oven using Agilent's recommended hybridization chamber. Subsequently, the microarrays were washed with the Agilent Gene Expression Wash Buffer 1 for 5 min at room temperature. A second washing step was performed with Agilent Gene Expression Wash Buffer 2 warmed to 37°C for 5min. Fluorescence signals after hybridization were detected with a DNA microarray scanner G2505C (Agilent Technologies) using one color scan setting for 8 × 60 K array slides (Scan Area 61 × 21.6 mm, Scan resolution 5 μm, Dye channel is set to Green and Green PMT is set to 100%).
4
Profiling miRNA Expression in Lung Cancer
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As in our previous work,21 miRNA expression profiles of lung cancer cells A549 and its DDP-resistant cells A549/DDP were analyzed by microarray. MiRNA expression profiles of exosomes derived from A549 and A549/DDP were also analyzed by microarray. The microarray experiment was performed by the BGI Company (Beijing, China). Exosomal RNA was extracted by Total RNA Purification Kit (Norgen, Thorold, ON, Canada) according to the manufacturer’s protocols, and total RNA of cells was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA, USA). RNA was quantified by NanoDrop® ND-2000 (Thermo Fisher Scientific, Waltham, MA, USA), and its quality was assessed by an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Human microRNA Microarray Kit (Agilent Technologies) was used for labeling and hybridization according to its instructions. The expression profiling of miRNA was conducted using the Agilent Human 8×60 K. The differential expression of miRNAs was screened to exclude those <1.5-fold changes in contrast to A549 or A-exo (exosomes of A549). The microarray data have been submitted to the Gene Expression Omnibus and the data could be accessed by the accession numbers GSE85603 and GSE85604.
5
Differential miRNA Expression in CD44+ and CD44- MKN45 Cells
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Total RNA from CD44(+) and CD44(−) MKN45 cells was prepared using TRIzol reagent according to the manufacturer's instructions. Microarrays were carried out using the Agilent Human miRNA 8 X 60K (Rel 16.0 V2) platform (Agilent Technologies, Palo Alto, CA, USA). RNA hybridizations were done with the Human microRNA Microarray Kit (Agilent Technologies) according to the manufacturer's protocol. Arrays were scanned on an Agilent C scanner. Images were quantified and data were processed using Agilent Feature Extraction Software (v 10.10.1.1). Raw data were extracted using software provided by the Agilent Feature Extraction Software (v 10.7.1.1). Selected miRNAgTotalGeneSignal values were logarithmically transformed and normalized by the quantile method. Each comparative analysis between test sample and control sample was carried out using fold‐change. Hierarchical cluster analysis was carried out using complete linkage and Euclidean distance as a measure of similarity.
6
Serum miRNA Profiling Using Microarray
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Frozen serum samples were thawed at room temperature. Total RNA was extracted from 500 μL of serum using TRIzol LS Reagent (Life technologies, Darmstadt, Germany) and the miRNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The RNA sample volume was reduced in a Speed Vac to a final volume of 1 μL and labeled and hybridized using the Human microRNA Microarray Kit (Rel16.0, Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. The detailed hybridization protocol and raw data are provided in NCBI's Gene Expression Omnibus (GEO, Series accession number GSE57570). For statistical analysis, signal intensities were normalized to the array median, log2 transformed and subjected to Student’s paired t-test. For heat map presentation, the Multiple Experiment Viewer tool version 4.9.0 (Dana-Farber Cancer Institute, Boston, MA, USA) was used. Prediction of miRNA targets was performed using TargetScan [29 (link)]. The predicted targets were classified in Gene Ontology categories of molecular function to estimate relevant biological processes. As background for the analysis, the probe set of the Agilent Whole Human Genome Oligo Microarray (8x60k) was chosen. Term enrichment relative to the expected background distribution was scored using Fisher’s Exact test with Benjamini–Hochberg correction.
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