Investigation Title Transcription profiling of Stratagene human universal reference RNA labelled with different labelling protocols to test labelling protocols Comment[Submitted Name] Variability in Microarray Labeling Methods Experimental Design optimization_design unknown_experiment_design_type co-expression_design transcription profiling by array Experimental Design Term Source REF mo mo EFO Comment[ArrayExpressReleaseDate] 2007-07-24 Comment[SecondaryAccession] GSE3254 Comment[AEMIAMESCORE] 5 Comment[SecondaryAccession] GDS1954 Comment[ArrayExpressAccession] E-GEOD-3254 Comment[MAGETAB TimeStamp_Version] 2010-10-15 05:21:34 Last Changed Rev: 14677 Experimental Factor Name Protocol Time Experimental Factor Type protocol time Experimental Factor Term Source REF Person Last Name Monzon Person First Name Federico Alberto Person Mid Initials Person Email monzonfa@upmc.edu Person Phone Person Fax Person Address Clinical Genomics Facility,Pathology,University of Pittsburgh,5230 Centre Ave., WG22,Pittsburgh,15232,USA Person Affiliation University of Pittsburgh Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2007-07-24 PubMed ID 16645204 Publication DOI 16645204 Publication Author List Changqing Ma, Maureen Lyons-Weiler, Wenjing Liang, William LaFramboise, John R Gilbertson, Michael J Becich, Federico A Monzon Publication Title In vitro transcription amplification and labeling methods contribute to the variability of gene expression profiling with DNA microarrays. Publication Status journal_article Publication Status Term Source REF The MGED Ontology Experiment Description Considerable variation in gene expression data from different DNA microarray platforms has been demonstrated. However, no characterization of the source of variation arising from labeling protocols has been performed. To analyze the variation associated with T7-based RNA amplification/labeling methods, aliquots of the Stratagene Human Universal Reference RNA were labeled using 3 eukaryotic target preparation methods and hybridized to a single array type (Affymetrix U95Av2). Variability was measured in yield and size distribution of labeled products, as well as in the gene expression results. All methods showed a shift in cRNA size distribution, when compared to un-amplified mRNA, with a significant increase in short transcripts for methods with long IVT reactions. Intra-method reproducibility showed correlation coefficients >0.99, while inter-method comparisons showed coefficients ranging from 0.94 to 0.98 and a nearly two-fold increase in coefficient of variation. Fold amplification for each method was positively correlated with the number of present genes. Two factors that introduced significant bias in gene expression data were observed: a) number of labeled nucleotides that introduces sequence dependent bias, and b) the length of the IVT reaction that introduces a transcript size dependent bias. This study provides evidence of amplification method dependent biases in gene expression data. Protocol Name P-G3254-5 P-G3254-4 P-G3254-2 P-G3254-3 P-G3254-1 Affymetrix:Protocol:Hybridization-EukGE-WS2v4 P-AFFY-6 Protocol Type labeling labeling labeling labeling labeling hybridization feature_extraction Protocol Description The Affymetrix GeneChip® Expression 3’-amplification Reagents for IVT labeling kit is used. However, the length of the in vitro transcription reaction is truncated to 4 hours. Therefore, it is called the 'Affy4h' method in the paper. Approximately 1 Âμg of total RNA was processed to produce biotinylated cRNA targets. UTP-biotin was used for labeling and the in vitro transcription reaction lasts for 4 hours. The Enzo BioArray High Yield™ RNA Transcript Labeling Kit is used. It is called the 'Enzo' method in the paper. Approximately 5 Âμg of total RNA was processed to produce biotinylated cRNA targets. UTP-biotin and CTP-biotin were used for labeling and the in vitro transcription reaction lasts for 4 hours. Affymetrix GeneChip® Expression 3’-amplification Reagents for IVT labeling kit is used. It is called the 'Affy' method in the paper. Approximately 1 Âμg of total RNA was processed to produce biotinylated cRNA targets. UTP-biotin was used for labeling and the in vitro transcription reaction lasts for 16 hours. The CodeLink Expression Assay Reagent Kit (GE Healthcare, Piscataway, NJ) is used. It is called the 'CodeLink' method in the paper. Approximately 1 Âμg of total RNA was processed to produce biotinylated cRNA targets. UTP-biotin was used for labeling and the in vitro transcription reaction lasts for 14 hours. The Affymetrix GeneChip® Expression 3’-amplification Reagents for IVT labeling kit is used. It is called the 'Affy' method in the paper. Approximately 1 Âμg of total RNA was processed to produce biotinylated cRNA targets. UTP-biotin was used for labeling and the in vitro transcription reaction lasts for 16 hours. Title: Fluidics Station Protocol. Description: Title: Affymetrix CEL analysis. Description: Protocol Parameters Protocol Hardware Protocol Software MicroArraySuite 5.0 MicroArraySuite 5.0 Protocol Contact Protocol Term Source REF SDRF File E-GEOD-3254.sdrf.txt Term Source Name The MGED Ontology ArrayExpress mo EFO The MGED Ontology Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version