Comment[ArrayExpressAccession] E-MTAB-3349 MAGE-TAB Version 1.1 Investigation Title Functional analysis of the Nkx2.2 SD domain: gene expression profiling of E16.5 Nkx2.2 SD mutant pancreata versus wild-type littermate pancreata Comment[Submitted Name] Functional analysis of the Nkx2.2 SD domain: gene expression profiling of E16.5 Nkx2.2 SD mutant pancreata versus wild-type littermate pancreata Experiment Description The experiment consists of 7 one channel assays for analyzing embryonic pancreas at E16.5. There are three or four replicates per condition. At E16.5, we are looking at differential gene expression across two mouse lines, WT vs Nkx2.2 SD mutants. Experimental Design genetic modification design Comment[AEExperimentType] transcription profiling by array Experimental Factor Name genotype Experimental Factor Type genotype Quality Control Type biological replicate Quality Control Term Source REF EFO Public Release Date 2015-01-29 Person Last Name Sussel Levine Person First Name Lori Joshua Person Mid Initials Person Email lgs2@columbia.edu jal2108@columbia.edu Person Phone 215-851-5232 Person Address 1150 St Nicholas Ave, NY, NY 10032 1150 St nicholas Ave, Room 605, NY, NY 10032 Person Affiliation Columbia University Columbia University Person Roles submitter investigator PubMed ID Publication Author List Publication Title Publication Status Protocol Name P-MTAB-43469 P-MTAB-43470 P-MTAB-43471 P-MTAB-43472 P-MTAB-43473 P-MTAB-43474 P-MTAB-43475 P-MTAB-43476 P-MTAB-43477 P-MTAB-43478 Protocol Type dissection protocol nucleic acid extraction protocol linear_amplification nucleic acid labeling protocol nucleic acid hybridization to array protocol array scanning protocol feature_extraction flag_filter within_bioassay_data_set_function normalization data transformation protocol Protocol Description Timed-pregnant mice were sacrificed using CO2 asphyxiation followed by cervical dislocation in accordance with our Columbia University IACUC approved protocol. Pancreata were removed from the embryos under a dissection microscope and immediately stored in RNALater (Ambion) at 4 C until genotypes were ascertained and RNA extraction was performed. Cells are extracted or tissue is dissected. This biological sample is then lysed and homogenized in the presence of a highly denaturing guanidine isothiocyanate (GITC)-containing buffer, which immediately inactivates RNases to ensure isolation of intact RNA. Ethanol is added to provide appropriate binding conditions, and the sample is then applied to an RNeasy column where the total RNA binds to the membrane and contaminants are efficiently washed away. High-quality RNA is then eluted in water. The RNeasy procedure represents a novel technology for RNA isolation. This technology combines the selective binding properties of a silica-gel-based membrane with the speed of microspin technology. A specialized high-salt buffer system allows RNA to bind to the RNeasy silica-gel membrane. This system uses the rapid and sensitive Ribo-SPIA TM RNA amplification process, which involves a series of enzymatic reactions resulting in linear amplification of exceedingly small amounts of RNA for use in array analysis. Unlike exponential RNA amplification methods, such as NASBA and RT-PCR, Ovation amplification maintains representation of the starting mRNA population. The amplification process resulted in a yield of 6-10 ug of amplified cDNA, which was purified using the QIAquick PCR Purification Kit (Qiagen, CA) and then eluted in coupling buffer (0.1M Sodium Bicarbonate, Ph 9). 2 ug of cDNA was mixed with Random Primers and denatured at 95 degrees for 5 min, then cooled briefly on ice. Next, the appropriate Cyanine dUTP fluorescent nucleotides (GE) were added, along with the nucleotide mix and Exo Klenow fragment. This was gently mixed and incubated at 37 degrees for 2 hours. The labeled samples were purified using the MinElute PCR Purification Kit (Qiagen, CA), and the efficiency of dye incorporation and yield was determined using the NanoDrop ND-1000 UV-Vis Spectrophotometer. 1.65 ug of cDNA was hybridized to the Agilent Whole Mouse Genome Microarray 4x44K [G4122F] for 17 hrs at 65 degrees. NA Agilent Feature Extraction Software allows extraction of feature intensities from Agilent and non-Agilent 1x3 glass slide microarrays with options to background correct, flag outliers, and normalize. Software version 10.5.1.1. The dataset was filtered to remove positive control reporters, any reporter that had been flagged as bad, reporters flagged as ABSENT in all conditions of the assay group, and reporters which were NA in 50% of the replicates in any condition. A threshold equal to the median + 2 IQR of the negative controls on the assay is computed. Then for each reporter whose intensity is less than the threshold, its intensity is reset to the threshold value. Quantile normalization with R bioconductor function normalizeBetweenArrays from the limma package Protocol Term Source REF EFO EFO MGED EFO EFO EFO MGED MGED MGED EFO Protocol Parameters foreground measurement;background measurement method;assay group Protocol Hardware Agilent DNA Microarray Scanner Model # G2505C Term Source Name EFO ArrayExpress MGED Term Source File http://www.ebi.ac.uk/efo http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php Comment[AdditionalFile:tif] FGC_251486829213_S01.tif Comment[AdditionalFile:tif] FGC_251486829214_S01.tif Comment[AdditionalFile:tif] FGC_251486829215_S01.tif Comment[AdditionalFile:tif] FGC_251486829216_S01.tif SDRF File E-MTAB-3349.sdrf.txt