Unknown,Transcriptomics,Genomics,Proteomics

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MicroArray analysis of miRNA expression of human esophageal cell samples and breast cell samples for comparison with multiplexed ligation-based miRNA detection assay

ABSTRACT: This experiment was carried out as part of a benchmarking study for Ligo-miR EZ, a ligation based multiplex miRNA detection assay. Differential analysis was performed for miRNA expression among the cell samples tested with the microarray and Ligo-miR EZ assay to confirm that Ligo-miR EZ gave the same results as an established, commercially available array. Primary, normal, non-immortalized human esophageal epithelial cells (HEEPIC), along with esophageal cancer cell lines (SKGT4 and OE33), were purchased from ScienCell Research Laboratories (Carlsbad, California, USA) and Sigma Chemical (St Louis, Missouri, USA), respectively. The Barrettâs esophageal cell lines (CHTRT and QHTRT) were gifts of Dr. Peter Rabinovitch, Fred Hutchinson Cancer Center. HEEPIC and CHTRT, QHTRT cells were cultured in EpiCM-2 medium. SKGT4 and OE33 were cultured with DMEM with 10% fetal bovine serum (FBS). Breast cell lines (MCF-7, MCF-10A, and MDA-MB-231) were purchased from ATCC (Manassas, VA). MCF-7 and MDA-MB-231 cells were grown in DMEM with 4.5 g/L glucose without L-glutamine and sodium pyruvate supplemented with 5 ÂµL/mL penicillin/streptomycin, 1% non-essential amino-acids, and 10% FBS. MCF-10A cells were grown in DMEM/F12 supplemented with 100 Âµg/mL epithelial growth factor (EGF), 10 mg/mL insulin, 1 mg/mL cholera toxin, 1 mg/mL hydrocortisone, 5 ÂµL/mL penicillin/streptomycin, and 5% horse serum. Total RNA was isolated from the cell lines using RNeasy kits (Qiagen, Valencia, CA), combined with RNase-free DNase (Qiagen, #79254), with TRIzol reagent (Life Technologies, Carlsbad, CA) used instead of the QIAzol. The total RNA was pooled before analysis to ensure that identical samples were used for each analysis method. Microarray analysis was performed using Agilent Human miRNA Microarray Kit Release 19.0, 8x60K (G4872A, Agilent Technologies, Santa Clara, CA) following manufacturerâs protocols. Quality checks of both total RNA and small RNA were performed using a 2100 Bioanalyzer and software which detect 28S and 18S ribosomal RNA ratio, total RNA Integrity Number (RIN), small RNA and miRNA concentrations in the total RNA isolated. Only samples with 28S/18S>1.2, RIN>8 and detectable miRNA were used for the study. 150 ng of total RNA were first dephosphorylated with 11.2 units of calf intestine alkaline phosphatase at 37Â°C for 30 minutes and followed by end-labeling with pCp-Cy3 and 15 units of T4 RNA ligase using miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, CA) at 16Â°C for 2 hours. Labeled samples were purified with Micro Bio-Spin 6 columns (Bio-Rad, Hercules, CA). Labeling efficiency and nucleic acid concentration were measured using Nanodrop 1000. Samples were then mixed with 10x blocking agent and 2x Hi-RPM hybridization buffer (Agilent Technologies) and hybridizations were carried out at 55Â°C with rotation at 20 rpm in a designated Agilent G2545A hybridization oven for 20 hours. Finally, microarrays were washed and scanned using an Agilent scanner controlled by Agilent Scan Control 7.0 software. Data were acquired with Agilent Feature Extraction 9.5.3.1 software for miRNA microarray. Two data files are generated for each array: Feature Extraction file contains signal intensities from all individual probes and GeneView file contains summarized signal intensities for each miRNA by combining intensities of replicate probes and background subtraction. Data normalization and analysis were performed using GeneSpring GX 11 following software developerâs recommendation (Agilent). miRNA signal intensities from GeneView files were imported into software. Signal intensity from each array was quantile normalized, and all negative intensity values were surrogated to 1 before analysis.

ORGANISM(S): Homo sapiens

SUBMITTER: John Kilburn

PROVIDER: E-MTAB-5575 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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MicroArray analysis of miRNA expression of human esophageal cell samples and breast cell samples for comparison with multiplexed ligation-based miRNA detection assay

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