Investigation Title A 50-Gene Predictor of Recurrence as a Guide to Therapy in Early-Stage Colon Cancer. Comment[Submitted Name] A 50-Gene Predictor of Recurrence as a Guide to Therapy in Early-Stage Colon Cancer. Experimental Design transcription profiling by array Experimental Design Term Source REF EFO Comment[SecondaryAccession] GSE10402 Comment[ArrayExpressReleaseDate] 2010-05-15 Comment[AEMIAMESCORE] 3 Comment[Publication DOI] 10.1073/pnas.0806674105 Comment[ArrayExpressAccession] E-GEOD-10402 Comment[MAGETAB TimeStamp_Version] 2010-07-29 23:26:56 Last Changed Rev: 13058 Experimental Factor Name AGE Experimental Factor Type age Experimental Factor Term Source REF Person Last Name Potti Acharya Ried Garman Acharya Nevins Sud Walters Grade Hsu Barry Ginsburg Mukerjee Person First Name Anil Chaitanya Thomas Katherine Chaitanya Ramanuj Joseph Shivani Kelli Marian David William Geoffrey Sayan Person Mid Initials R S R S S T S Person Email c.acharya@duke.edu Person Phone Person Fax Person Address Potti Lab, IGSP, Duke University Medical Center, 101 Science Dr, Rm#2211, Durham, 27705, USA Person Affiliation Duke University Medical Center 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 2010-05-15 PubMed ID 19050079 Publication DOI 19050079 Publication Author List Garman KS, Acharya CR, Edelman E, Grade M, Gaedcke J, Sud S, Barry W, Diehl AM, Provenzale D, Ginsburg GS, Ghadimi BM, Ried T, Nevins JR, Mukherjee S, Hsu D, Potti A Publication Title A genomic approach to colon cancer risk stratification yields biologic insights into therapeutic opportunities. Publication Status journal_article Publication Status Term Source REF The MGED Ontology Experiment Description Gene expression profiles reflect unique aspects of individual biologic phenotypes and may characterize the heterogeneity of solid tumors. Using previously-described methodologies that employ DNA microarray data, a 50-gene expression profile (metagene) that predicts risk of recurrence in early stage colon carcinoma was identified. This analysis used an initial discovery cohort of 52 patients. The performance of the 50-gene predictor was evaluated in an independent validation cohort of 73 patients. Using a connectivity map analysis of the 50-gene model, we identified candidate agents and then tested the in vitro efficacy of these compounds in colon cancer cell lines. 73 samples that had patient recurrence data with stage information were used in the analysis. Keywords: Disease state analysis A total of 73 samples were spotted on microarray slides. No replicates are included in the study. Protocol Name P-GSE10402-2 P-GSE10402-3 P-GSE10402-4 P-GSE10402-5 P-GSE10402-6 P-GSE10402-7 P-GSE10402-1 Protocol Type nucleic_acid_extraction labeling hybridization hybridization image_aquisition feature_extraction bioassay_data_transformation Protocol Description Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Cy3 and Cy5 Labeling protocols - generic NCI Oligo Microarray Hybridization Other: For pre-hybrization, apply 40 ul of pre-hybridization buffer (5X SSC, 0.1% SDS, 1% BSA) to the array and incubate at 42øC for at least 30 minutes and up to an hour. Wash off the pre-hybridization solution by rapidly plunging the slide in distilled water for 2 minutes, then transfer slide to 100% isopropanol for 2 minutes. Allow slide to air dry completely prior to use. (Can spin dry if in a rush.) (NOTE: Do not exceed 1 hour after pre-hybridization/drying before setting up hybridization.) For hybridization, combine Cy3 and Cy5 labeled targets together (~9 ul recovered for each). Add 1 ul COT-1 DNA (8-10 ug/ul) and 1 ul poly A (8-10 ug/ul). Denature target at 100øC for 1 minute, then snap cool on ice. (Final volume should be about 20 ul.) Make fresh 2X Formamide hybridization buffer (50% formamide, 10X SSC, 0.2% SDS) and warm to 42øC just before adding to samples. Add 20 ul of 2X F-hyb buffer to samples. Load 40 ul sample onto microarray. Add 20 ul of 3X SSC to wells in hyb chamber to maintain humidity. Incubate overnight (12-16 hours) at 42øC in water bath or hybridization oven. After hybridization of slides, wash slides for 2 minute in 2X SSC with 0.1% SDS (with occasional plunging), for 2 minute in 1X SSC (with occasional plunging), for 2 minutes in 0.2X SSC (with occasional plunging), and spin for 3 minutes at 650 rpm to dry. (Refer to NCI Microarray Manual) Slides were scanned on an Axon scanner using GenePixPro (3.0) software. Spot quality was assessed according to criteria in GenePixPro (3.0) software. Background subtraction and normalization was performed upon data extraction from the CIT/NIH-microarray database, mAdb (http://nciarray.nci.nih.gov/). After background correction and removal of flagged values, log base 2 expression ratios were median centered and linear transformed to obtain the log and linear values given in the data table. ID_REF =
VALUE = lowess normalized log2 ratio Protocol Parameters Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF The MGED Ontology The MGED Ontology SDRF File E-GEOD-10402.sdrf.txt Term Source Name The MGED Ontology ArrayExpress EFO The MGED Ontology Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version