Unknown,Transcriptomics,Genomics,Proteomics

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A wholly-defined Agilent microarray spike-in dataset

ABSTRACT: We have generated a wholly defined spike-in dataset for Agilent microarrays consisting of 12 arrays with more than 2000 differentially expressed, and approximately 3600 background, cRNAs. The composition of this âAg Spikeâdataset is identical to that of our previous Platinum Spike dataset (GSE21344) and therefore allows direct cross-platform comparison. Comparison between the Ag Spike and Platinum Spike studies shows high agreement between results obtained using the Affymetrix and Agilent platforms. We used the same 28 PCR pools that had been used before to generate the Platinum-Spike dataset (GSE21344). These pools contain PCR products from 5725 Drosophila Gene Collection release 1.0 (DGCr1) cDNA clones. We mixed PCR products from all pools together with specified relative abundance to generate two samples representing the A and B conditions. Both samples were in vitro transcribed and then labeled with Cy3 and Cy5 fluorescent dye. Labeled A samples were hybridized with B samples labeled with reverse dye to three Agilent Drosophila Gene Expression microarrays. The process from in vitro transcription to hybridization was repeated twice. At the end we generated 12 arrays that were randomly placed in three independent slides to remove potential systematic error due to array position.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Qianqian Zhu

PROVIDER: E-GEOD-24866 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The most widely-used method for detecting genome-wide protein-DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms and analysis algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and "spike-ins" comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups. Blind to the number of spike-ins, their locations, and the range of concentrations, each group made predictions of the spike-in locations. All commercial tiling array platforms performed well, although each platform and analysis algorithm had distinct performance and cost characteristics. Simple sequence repeats and genome redundancy tend to result in false positives on oligonucleotide platforms. The spike-in DNA samples and the resulting array data presented here provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated. Keywords: chip-ChIP simulation For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf For each replicate array, we labeled 1Âµg of spike-in sample and 1Âµg of control sample. The Bioprime Plus Array CGH labeling Module (Invitrogen Catolog number 18095-014) was used. The spike-in was labeled with the red channel dye (Alexa Fluor-647) and the control was labeled with the green channel dye (Alexa Fluor-555) in two of the replicates. A dye swap was performed for the third replicate. These samples were then competitively hybridized to three replicate 244k arrays from Agilent, (AMADID 25150451). We hybridized the samples to the arrays using the Agilent Array CGH protocol, with some modifications. Briefly, labeled DNA was combined with Cot-1 DNA, blocking reagent, and hybridization buffer. The hybridizations were carried out at 60Â°C. The arrays were scanned using an Agilent dual laser scanner, and the images were processed using Agilent Feature Extraction Software.

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A wholly-defined Agilent microarray spike-in dataset

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