Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling validation of an custom mouse oligo array using liver samples following treatment with the hepatotoxin phenobarbital and variable hybridization protocols

ABSTRACT: The use of custom-made focused microarrays has become a popular alternative, allowing the study of genes relevant to a specific hypothesis. To address the limitations of commercial arrays in toxicogenomics investigations, we have developed a custom mouse oligonucleotide microarray, the HC ToxArrayTM, which, by virtue of including genes that respond to a variety of chemical and physical stressors, is more relevant for toxicological studies. Furthermore, we have incorporated an extensive series of controls useful for both quality control and normalization of small focused arrays. Validation of the EC series for normalization was achieved by determining the gene expression profile in liver samples following treatment with the hepatotoxin phenobarbital. Technical variability experiments were also carried out by two different technicians using two hybridization methods (automated and manual) and compared. In this study, we demonstrate that dilution of oligonucleotides on the microarray itself provides an innovative approach allowing the full dynamic range of the scanner to be covered with a single gene spike-in. The dilution series can be used in a composite normalization to improve detection of differential gene expression and provide quality control measures.

INSTRUMENT(S): ScanArray Express HT [PerkinElmer]

ORGANISM(S): Mus musculus

DISEASE(S): normal

SUBMITTER: Iain Lambert

PROVIDER: E-MEXP-525 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Novel design and controls for focused DNA microarrays: applications in quality assurance/control and normalization for the Health Canada ToxArray.

Yauk Carole L CL Yauk Carole L CL Williams Andrew A Boucher Sherri S Berndt Lynn M LM Zhou Gu G Zheng Jenny L JL Rowan-Carroll Andrea A Dong Hongyan H Lambert Iain B IB Douglas George R GR Parfett Craig L CL

BMC genomics 20061019

<h4>Background</h4>Microarray normalizations typically apply methods that assume absence of global transcript shifts, or absence of changes in internal control features such as housekeeping genes. These normalization approaches are not appropriate for focused arrays with small sets of genes where a large portion may be expected to change. Furthermore, many microarrays lack control features that can be used for quality assurance (QA). Here, we describe a novel external control series integrated w ...[more]

PMID: 17052352

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Project description:In this study, we employ high-density oligonucleotide microarrays to characterize the MutaMouse FE1 cell line at various stages of cell growth, in primary MutaMouse lung epithelial cell cultures, and in whole lung. Global transcriptional analysis and real-time RT-PCR was applied to (1) further define the cellular origin of the FE1 cell line and its responses under different culture conditions (media and substratum), (2) provide insight into the transcriptional differences in cellular processes between FE1 cultures compared to whole lung tissues, more specifically in toxicological response, and (3) preliminarily examine FE1 culture response to exposure of benzo(a)pyrene compared to whole animals. Total RNA samples from 3 cell culture types (50% FE1, %100 FE1, and Primary lung) or MutaMouse lung were labeled with Cyanine 5-CTP, and universal reference total RNA (Stratagene, CA, USA) was labeled with Cyanine 3-CTP (Perkin Elmer Life Sciences, Woodbridge, ON, Canada) using Agilent linear Amplification kits (Agilent Tech. Inc. Mississauga, ON, Canada) following the manufacturer's instruction. Briefly, double-stranded cDNA was synthesized using MMLV-RT with T7 promoter primer, starting with 5 ug total RNA. Cyanine-labeled cRNA targets were in vitro transcribed using T7 RNA polymerase. The synthesized cRNA was precipitated by LiCl and fragmented at 60 degrees C for 30 min with fragmentation solution. Cy5- sample cRNA and Cy3- reference cRNA were hybridized to Agilent mouse development microarrays (containing ~20,000 unique 60 mer oligonucleotides; Agilent Tech. Inc. Mississauga, ON, Canada) at 60 degrees C overnight with Agilent hybridization solution and washed according to manufacturer's instruction. Arrays were scanned on a VersArray ChipReader (BioRad Laboratories Ltd., Waterloo, Ontario, Canada), and data were acquired with ImaGene 5.5 (BioDiscovery, Inc. CA, USA). Present calls were determined as signals that were greater than the mean plus three times the standard deviation of the average of the negative control spots.

Dataset Information

Transcription profiling validation of an custom mouse oligo array using liver samples following treatment with the hepatotoxin phenobarbital and variable hybridization protocols

Publications

Novel design and controls for focused DNA microarrays: applications in quality assurance/control and normalization for the Health Canada ToxArray.

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