Comment[ArrayExpressAccession] E-MTAB-2715 MAGE-TAB Version 1.1 Investigation Title Affymetrix MOE430v2 vs Mouse PancChip 5.0 comparison Comment[Submitted Name] Affymetrix MOE430v2 vs Mouse PancChip 5.0 comparison Experiment Description Comparison of two microarray platforms: the Mouse PancChip 5.0 and the Affymetrix GeneChip Mouse Genome 430 2.0 Array. The aim of this experiment was to determine the ability to identify differentially expressed genes in islet and pancreas RNA, and the sensitivity of the two platforms, using the same source material in a carefully controlled manner. RNA was extracted from adult mouse pancreas (n=5) and highly purified islet samples (n = 5). All samples were amplified once. 5 biological replicates (islets vs. pancreas) in a dye swap experimental design were hybridized to the PancChip. 3 biological replicates, using the same amplified RNA hybridized to the PancChip, of both the pancreas and islet samples were also hybridized to the GeneChip. All data were normalized using appropriate methods and differential expression between islet and pancreas was determined using PaGE with a 10% FDR. The study revealed that the PancChip is a highly cost effective alternative to the Affymetrix 430-2, that the PancChip is up to 60% more sensitive than the Affymetrix 430-2 GeneChip and that 80% (7,000) of the probe sets on the PancChip show expression in either Islets or Pancreas, while only 25% (6,800) show expression on the Affymetrix GeneChip. Experimental Design organism part comparison design array_platform_comparison_design dye swap design Comment[AEExperimentType] transcription profiling by array Experimental Factor Name individual array design organism part Experimental Factor Type individual array design organism part Quality Control Type biological replicate dye-swap quality control role Quality Control Term Source REF EFO EFO Public Release Date 2014-07-01 Person Last Name Kaestner Manduchi Stoeckert Brestelli Person First Name Klaus Elisabetta Christian John Person Mid Initials J. E. Person Email kaestner@mail.med.upenn.edu manduchi@upenn.edu stoeckrt@upenn.edu jbrestel@upenn.edu Person Phone 215-898-8759 215-573-4408 215-573-4409 Person Address 12-126 SCTR, 3400 Civic Center Blvd, Philadelphia, PA 19104-6145, USA 1428 Blockley Hall, 423 Guardian Drive, Philadelphia, PA 19104-6021, USA 1415 Blockley Hall, 423 Guardian Drive, Philadelphia, PA 19104-6021, USA Person Affiliation University of Pennsylvania University of Pennsylvania University of Pennsylvania University of Pennsylvania Person Roles submitter data analyst investigator experiment performer Protocol Name P-MTAB-39905 P-MTAB-39906 P-MTAB-39907 P-MTAB-39908 P-MTAB-39909 P-MTAB-39910 P-MTAB-39911 P-MTAB-39912 P-MTAB-39913 P-MTAB-39914 P-MTAB-39915 P-MTAB-39916 P-MTAB-39917 P-MTAB-39918 P-MTAB-39919 P-MTAB-39920 Protocol Type purify nucleic acid extraction protocol linear_amplification split nucleic acid labeling protocol nucleic acid labeling protocol hybridization hybridization image_acquisition image_acquisition feature_extraction feature_extraction feature_extraction loess_global_normalization flag_filter dye_swap_merge Protocol Description Mouse islets were isolated after collagenase treatment and purification over a ficoll gradient. Tissue was quickly removed and immediately homogenized in 10 ml denaturing solution (4 M guanidium thiocyanate, 0.1 M Tris-Cl pH 7.5, 1% beta-mercaptoethanol). Total RNA was extracted using an acid-phenol extraction procedure. Following chlorophorm extraction, 1 volume 70% EtOH was added to the aqueous phase and applied to RNeasy column (Qiagen). Extraction followed manufacturer's recommendations. RNA is amplified using the MessageAmp aRNA Kit (Ambion, Austin, TX). Total RNA is reverse transcribed using a T7 Oligo(dT) Primer and reverse transcriptase at 42C for 2 hours. Second-strand synthesis is performed using DNA Polymerase. The resulting cDNA is purified with a filter cartridge. Double-stranded cDNA is concentrated and in vitro transcribed at 37 C to make antisense RNA (aRNA). The aRNA is DNase-treated, purified with a filter cartridge, and eluted with nuclease-free water. aRNA aliquoting. Amplified RNA and random hexamers are brought to 25 ul and denatured for 5 min at 70 C. The reaction is then cooled to 42 C and an equal volume of RT reaction mix, 400 U SuperScript II, modified nucleotides, and 20 U RNasin is added. After appropriate incubation, the reaction is denatured, then neutralized, the buffer removed, and the cDNA dried. Dyes are coupled to the cDNA in the dark at room temperature (1 h). The reaction is then quenched. The aRNA was reverse transcribed using a T7 Oligo (dT) primer in order to obtain cDNA with a T7 promoter. Following cDNA symthesis, the aRNA went through a second round of amplification to obtain the yields required for a proper Affymetrix experiment. 20ug cRNA was submitted to the Penn Microarray Facility where it was futher Biotin-labeled. Slides are Pre-Hybridized in 5X-SSC, 0.1% SDS , 1% BSA at 42 C for 45 min. Rinsed 5X in deionized H20 and 1X in Isopropanol and dried by centrifugation for 1 min at 1000rpm. The cDNA hybridization mix was combined with mouse Cot-1 DNA and T7-Oligo dT buffers and incubated overnight at 42 C in a Corning hybridization chamber. Coverslip removed in 2X SSC, 0.1%SDS at room temp. Slide was washed once in 0.2X SSC, 0.1% SDS (42 C, 5 min, with agitation), once in 0.2X SSC (room temperature, 5 min, with agitation)and dried by centrifugation at 1000rpm for 3 min. The fragmented cRNA is mixed with the hybridization cocktail, and heat shocked at 99C for 5 minutes. In the meanwhile, the probe array is filled with hybridization buffer and incubated at 45C for 10 minutes with rotation. The heated hybridization cocktail is then transferred to a 45C heat block for 5 minutes, and spun at maximum speed in a microcentrifuge for 5 minutes. Fill the probe array cartridge with the clarified hybridization cocktail and place probe array in rotisserie box in 45C oven. Hybridize for 16 hours. The Affymetrix GeneChip Scanner 3000 is a wide-field, epifluorescent, near-confocal microscope. The scanner uses a 532 nm solid-state laser to excite probe array fluorophores. This in turn produces an emission wavelength at about 570 nm. As the array is scanned, a photomultiplier tube collects and converts the fluorescent emissions into an electrical signal, which is converted by an analog-digital converter into corresponding numeric values representative of fluorescent intensities. Agilent DNA Microarray Scanner Model #G2565BA The Cell Analysis algorithm calculates a single intensity value for each probe cell on an array. This intensity value, representative of the hybridization level of its target, is derived as follows. The bordering pixels of the probe cell are excluded. The remaining pixel intensity distribution is calculated, and the intensity value associated with 75% of the distribution is used as the probe cell intensity. The intensities for all probe cells are saved in the .CEL file. An absolute expression analysis examines the cell intensity file (*.CEL) from one experiment. For each transcript represented on the probe array, the expression algorithm computes the 1) detection call [present, absent, or marginal (unable to call the transcript present or absent), or no call]; 2) detection p-value; 3) signal (background subtracted and adjusted for noise); 4) stat pairs; 5) stat pairs used. The expression values are saved in the .CHP file. GenePix quantification From http://www.bioconductor.org. Filter out spots with a given value for the GenePix flag measurement. See page 63 of http://files.axon.com/downloads/manuals/GenePix_Pro_6.0_User_Guide_Rev_K.pdf. Dye-swap merging of M (=log(Ch1)-log(Ch2)) values takes as input, for each spot, its values say M and M', from a pair of dye-swap 2-channel assays and outputs (M-M')/2. Protocol Term Source REF MGED EFO MGED MGED EFO EFO MGED MGED MGED MGED MGED MGED MGED MGED MGED MGED Protocol Hardware Affymetrix Fluidics Station 400 Affymetrix GeneChip Scanner 3000 (GCS3000) Agilent DNA Microarray Scanner Model #G2565BA Protocol Parameters T7-oligo-dT Amount;Oligo dT(21) Amount;Mouse Cot-1 DNA Amount Fluidics_Station;Fluidics_Module;Fluidics_Protocol Filter;Number of Scans; Pixel Size laser power;pmt Alpha1;Alpha2;SF;TGT;Tau background density measure;ratio formulations;software version;standard deviation R version;marray version;foreground_measurement;background_measurement;numerator_channel;denominator_channel;spots used for loess curve;smoothing_param;mapping_file flag_value numerator;denominator 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] 12996071_Cy3.tif 12996074_Cy3.tif 12996079_Cy3.tif 12996080_Cy3.tif 12996081_Cy3.tif 12996082_Cy3.tif 12996083_Cy3.tif 12996084_Cy3.tif 12996085_Cy3.tif 12996090_Cy3.tif Comment[AdditionalFile:tif] 12996071_Cy5.tif 12996074_Cy5.tif 12996079_Cy5.tif 12996080_Cy5.tif 12996081_Cy5.tif 12996082_Cy5.tif 12996083_Cy5.tif 12996084_Cy5.tif 12996085_Cy5.tif 12996090_Cy5.tif Comment[AdditionalFile:DAT] K1_MOE430v2.DAT K2_MOE430v2.DAT K3_MOE430v2.DAT K4_MOE430v2.DAT K5_MOE430v2.DAT K6_MOE430v2.DAT SDRF File E-MTAB-2715.sdrf.txt