ArticleThe Journal of molecular diagnostics : JMD2006
In vitro transcription amplification and labeling methods contribute to the variability of gene expression profiling with DNA microarrays.
Article in The Journal of molecular diagnostics : JMD, 2006. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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13 citing papers in PubMed.
- Controlling technical variation amongst 6693 patient microarrays of the randomized MINDACT trial.Communications biology · 2020Article
- AffyRNADegradation: control and correction of RNA quality effects in GeneChip expression data.Bioinformatics (Oxford, England) · 2013Article
- Development of the "Three-step MACS": a novel strategy for isolating rare cell populations in the absence of known cell surface markers from complex animal tissue.Journal of biomolecular techniques : JBT · 2012Article
- Estimating RNA-quality using GeneChip microarrays.BMC genomics · 2012Article
- Influence of RNA labeling on expression profiling of microRNAs.The Journal of molecular diagnostics : JMD · 2012Article
- Quality assurance of RNA expression profiling in clinical laboratories.The Journal of molecular diagnostics : JMD · 2012Review
- An adaptable method using human mixed tissue ratiometric controls for benchmarking performance on gene expression microarrays in clinical laboratories.BMC biotechnology · 2011Article
- A novel approach for reliable microarray analysis of microdissected tumor cells from formalin-fixed and paraffin-embedded colorectal cancer resection specimens.Journal of molecular medicine (Berlin, Germany) · 2009Article
- Motif composition, conservation and condition-specificity of single and alternative transcription start sites in the Drosophila genome.Genome biology · 2009Article
- Interlaboratory performance of a microarray-based gene expression test to determine tissue of origin in poorly differentiated and undifferentiated cancers.The Journal of molecular diagnostics : JMD · 2008Article
- The effect of column purification on cDNA indirect labelling for microarrays.Plant methods · 2007Article
- Comparison of target labeling methods for use with Affymetrix GeneChips.BMC biotechnology · 2007Article
- Gene expression profiles of prostate cancer reveal involvement of multiple molecular pathways in the metastatic process.BMC cancer · 2007Article
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7 authors.
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Abstract
The effect of different amplification and labeling methods on DNA microarray expression results has not been previously delineated. To analyze the variation associated with widely accepted T7-based RNA amplificationand labeling methods, aliquots of the Stratagene Human Universal Reference RNA were labeled using three eukaryotic target preparation methods followed by uniform replicate array hybridization (Affymetrix U95Av2). Method-dependent variability was observed in the yield and size distribution of labeled products, as well as in the gene expression results. A significant increase in short transcripts, when compared to unamplified mRNA, was observed in methods with long in vitro transcription reactions. Intramethod reproducibility showed correlation coefficients >0.99, whereas intermethod comparisons showed coefficients ranging from 0.94 to 0.98 and a nearly twofold increase in coefficient of variation. Fold amplification for each method positively correlated with the number of genes present. Our experiments uncovered two factors that introduced significant bias in gene expression data: the number of labeled nucleotides, which introduces sequence-dependent bias, and the length of the in vitro transcription reaction, which introduces transcript size-dependent bias. This study provides evidence that variability in expression data may be caused, in part, by differences in amplification and labeling protocols.
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