Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of rat peritoneal mesotheliomas following treatment with o-nitrotoluene or bromochloracetic acid

ABSTRACT: This study was performed to characterize the gene expression profile of rat peritoneal mesothelioma (RPM) formation following treatment of Fischer 344 rats with o-nitrotoluene (o-NT) or bromochloracetic acid (BCA). Experiment Overall Design: Four mesotheliomas from o-NT-treated rats and four mesotheliomas from BCA-treated rats were selected for microarray analysis. At necrosy, the tumors were collected, minced and flash frozen in liquid nitrogen. A non-transformed mesothelial cell line, Fred-PE (passage eight), was used as a source of reference RNA. Total RNAs from tumor tissue and Fred-PE were isolated with the RNeasy kit and 500 ng of total RNA was amplified and labeled using the Agilent Low RNA Input Fluorescent Linear Amplification Kit according to the manufacturer's protocol. For each two color comparison, 750 ng of each Cy3- and Cy5-labeled cRNA were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 17 hours in a rotating hybridization oven according to the Agilent 60-mer oligo microarray processing protocol prior to washing and scanning with an Agilent Scanner. Data was obtained using the Agilent Feature Extraction software (v7.5), using default for all parameters. Two arrays for each comparison were used, incorporating a fluor reversal.

ORGANISM(S): Rattus norvegicus

DISEASE(S): normal

SUBMITTER: NIEHS Microarray Group

PROVIDER: E-GEOD-4682 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Major carcinogenic pathways identified by gene expression analysis of peritoneal mesotheliomas following chemical treatment in F344 rats.

Kim Yongbaek Y Ton Thai-Vu TV DeAngelo Anthony B AB Morgan Kevin K Devereux Theodora R TR Anna Colleen C Collins Jennifer B JB Paules Richard S RS Crosby Lynn M LM Sills Robert C RC

Toxicology and applied pharmacology 20060207 2

This study was performed to characterize the gene expression profile and to identify the major carcinogenic pathways involved in rat peritoneal mesothelioma (RPM) formation following treatment of Fischer 344 rats with o-nitrotoluene (o-NT) or bromochloracetic acid (BCA). Oligo arrays, with over 20,000 target genes, were used to evaluate o-NT- and BCA-induced RPMs, when compared to a non-transformed mesothelial cell line (Fred-PE). Analysis using Ingenuity Pathway Analysis software revealed 169 c ...[more]

PMID: 16460773

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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare adhesion gene clusters and clock genes / biomarkers at different time points throughout the day in sorted murine lympahtic endothelial cells. Methods: RNA of sorted dermal LECs harvested in TriZol-LS was purified using Direct-zol RNA mini Prep Kit (ZymoResearch) following manufacturer’s instructions. Isolated RNA was quantified using the Nanodrop (Thermofisher) and analyzed on a Bioanalyzer (Agilent) using the RNA 6000 Nano or Pico Kit (Agilent). 75 ng of eluted total RNA was digested with DNase (Thermofisher) to remove DNA contaminations. An additional purification step with RNAClean XP Beads (Agencourt) was performed. The purified, bead-bound RNA was directly used as input in the SMART-Seq v4 Ultra Low Input RNA Kit (TaKaRA Bio). Full-length cDNA was generated following manufacturer’s instructions. Full-length cDNA was quantified using the Qubit dsDNA HS Assay Kit (Invitrogen) with the Qubit fluorometer. Finally, sequencing libraries were generated using 500 pg full-length cDNA following the NexteraXT protocol (Illumina). Libraries were quantified on a Bioanalyzer (Agilent) using the DNA 1000 Kit (Agilent) and sequenced on a HiSeq1500 system (Illumina) with a readlength of 100 nt, single-end mode. Results: obtained transcriptome profiles were processed on a Galaxy web interface hosted by LAFUGA (Gene Center, Munich). After demultiplexing and trimming data was mapped against the mouse genome (mm10) using RNA-STAR mapper (Galaxy Version 2.5.2b-0). Abundant reads were counted using HTSeq-count (Galaxy Version 1.0.0). Afterwards, gene expression analysis to detect differentially expressed genes was performed using DESeq2 (Galaxy Version 2.11.40.6), setting the false discovery rate (FDR) <0.05. Comparing the expression profiles and the enrichment of gene ontology cluster adhesion revealed a highly time-of-day dependent expression profile of genes. Conclusions: Our study represents the first detailed analysis of time-of-day dependent lymphatic endothelial cell transcriptomes, with respect to adhesion and clock genes. This will provide valid input into chronotherapy, timed delivery of substances and new therapeutic avenues.

Dataset Information

Transcription profiling of rat peritoneal mesotheliomas following treatment with o-nitrotoluene or bromochloracetic acid

Publications

Major carcinogenic pathways identified by gene expression analysis of peritoneal mesotheliomas following chemical treatment in F344 rats.

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