Explaining Differences in Saturation Levels for Affymetrix GeneChip Arrays
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE7110 | GEO | 2007/06/15
SECONDARY ACCESSION(S): PRJNA98415
REPOSITORIES: GEO
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Project description:The experimental spike-in studies of microarray hybridization conducted by Affymetrix demonstrate a nonlinear response of fluorescence intensity signal to target concentration. It was shown that the Langmuir adsorption isotherm recapitulates a general trend of signal response to concentration. However, this model fails to explain some key properties of the observed signal. In particular, according to the simple Langmuir isotherm, all probes should saturate at the same intensity level. However, this effect was not observed in the publicly available Affymetrix spike-in datasets. In our experimental study, we attempt to account for the unexplained variation in the observed probe intensities using customized fluidics scripts. We explore experimentally the effect of the stringent wash, target concentration, and hybridization time on the final microarray signal. Experiment Overall Design: The washing effect is assessed by scanning chips both prior to and after the stringent wash (Wash Condition). Selective labeling of both specific and non-specific targets allows the visualization and investigation of the washing effect for both specific and non-specific signal components. By varying the length of the hybridization cycle, the issue of attaining probe-target binding equilibrium can be addressed (Hybridization Time). To explore saturation potential, we performed hybridization experiments in which four clones are spiked-in and hybridized to two chips at a concentration of 1nM and 10nM respectively, each in the absence of complex background (Labeling Condition).
2008-06-15 | E-GEOD-7110 | biostudies-arrayexpress
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Project description:Transcript profiling is crucial to study biological systems and various platforms have been implemented to survey mRNAs at the genome scale. We have assessed the characteristics of the CATMA microarray designed for Arabidopsis thaliana transcriptome analysis, and compared it with two commercial platforms from Agilent and Affymetrix. The CATMA array consists of gene-specific sequence tags of 150 to 500 base pairs, the Agilent (Arabidopsis 2) array of 60mer oligonucleotides, and the Affymetrix gene chip (ATH1) of 25mer oligonucleotide sets. We have matched each probe repertoire with the Arabidopsis genome annotation (TIGR release 5.0) and determined the correspondence between them. Array performance was analyzed by hybridization with labeled target derived from eight RNA samples made of shoot total RNA spiked with a calibrated series of 14 control transcripts. A total of fourteen cDNA clones were thus selected and used as templates to synthesize bona fide polyadenylated spike RNAs. Each spike RNA was calibrated then mixed in equal amount with one of the other spike RNAs to obtain seven pairs at equal concentration. These seven spike RNA pairs were then combined systematically to construct seven complex spike mixes in a design similar to an ordered Latin square, each mix containing six of the seven spike pairs in staggered concentrations covering five logs. To prevent loss of spike RNA through adsorption to the plastic ware, the spike mixes were prepared in 0.5 µg/µl Col RNA, resulting in a range of concentration from 0.1 to 10,000 copies per cellular equivalent (cpc), assuming that the total RNA contained 1% polyadenalated mRNA and that a cell contained on average 300,000 transcripts. These seven RNA samples included equal amounts of combined spike RNA . To convert the spike hybridization signals to ratios, an eighth sample was prepared, called the reference sample, consisting of the base Col RNA completed with all spike RNAs at a concentration of 100 cpc. The results from the eight experiments using the Affymetrix gene chips (ATH1) are available for analysis or download from this site. Experimenter name = Jim Beynon Experimenter phone = 01798 470382 Experimenter fax = 01789 470552 Experimenter department = Horticulture Research International Experimenter institute = Warwick University Experimenter address = Horticulture Research International Experimenter address = Wellesbourne Experimenter address = Warwick Experimenter zip/postal_code = CV34 6QJ Experimenter country = UK Keywords: unknown_experiment_design_type
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Project description:Microarray measurements are susceptible to error caused by cross-hybridization and bulk hybridization. To characterize the effect of these off-target interactions, we analyzed samples made from baseline (S. cerevisiae) alone or mixed with a high-concentration 1kb spike. The spike is a 1 kb sequence amplified from the Schizosaccharomyces pombe gene SPAC27D7.10c, with an added oligo(dT)-T7 promoter. The baseline and spike samples were mixed at the double-stranded cDNA stage. Three arrays: 100% baseline, 99% baseline + 1% spike, 90% baseline + 10% spike. There are no replicates.
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Project description:Transcript profiling is crucial to study biological systems and various platforms have been implemented to survey mRNAs at the genome scale. We have assessed the characteristics of the CATMA microarray designed for Arabidopsis thaliana transcriptome analysis, and compared it with two commercial platforms from Agilent and Affymetrix. The CATMA array consists of gene-specific sequence tags of 150 to 500 base pairs, the Agilent (Arabidopsis 2) array of 60mer oligonucleotides, and the Affymetrix gene chip (ATH1) of 25mer oligonucleotide sets. We have matched each probe repertoire with the Arabidopsis genome annotation (TIGR release 5.0) and determined the correspondence between them. Array performance was analyzed by hybridization with labeled target derived from eight RNA samples made of shoot total RNA spiked with a calibrated series of 14 control transcripts. A total of fourteen cDNA clones were thus selected and used as templates to synthesize bona fide polyadenylated spike RNAs. Each spike RNA was calibrated then mixed in equal amount with one of the other spike RNAs to obtain seven pairs at equal concentration. These seven spike RNA pairs were then combined systematically to construct seven complex spike mixes in a design similar to an ordered Latin square, each mix containing six of the seven spike pairs in staggered concentrations covering five logs. To prevent loss of spike RNA through adsorption to the plastic ware, the spike mixes were prepared in 0.5 µg/µl Col RNA, resulting in a range of concentration from 0.1 to 10,000 copies per cellular equivalent (cpc), assuming that the total RNA contained 1% polyadenalated mRNA and that a cell contained on average 300,000 transcripts. These seven RNA samples included equal amounts of combined spike RNA . To convert the spike hybridization signals to ratios, an eighth sample was prepared, called the reference sample, consisting of the base Col RNA completed with all spike RNAs at a concentration of 100 cpc. The results from the eight experiments using the Affymetrix gene chips (ATH1) are available for analysis or download from this site. Experimenter name = Jim Beynon; Experimenter phone = 01798 470382; Experimenter fax = 01789 470552; Experimenter department = Horticulture Research International; Experimenter institute = Warwick University; Experimenter address = Horticulture Research International; Experimenter address = Wellesbourne; Experimenter address = Warwick; Experimenter zip/postal_code = CV34 6QJ; Experimenter country = UK Experiment Overall Design: 8 samples were used in this experiment
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