In vitro transcription amplification and labeling methods contribute to the variability of gene expression profiling with DNA microarrays.

The Journal of Molecular Diagnostics : JMD
Changqing MaFederico A Monzon

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 i...Continue Reading

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Citations

Dec 11, 2008·Journal of Molecular Medicine : Official Organ of the Gesellschaft Deutscher Naturforscher Und Ärzte·Silke LassmannMartin Werner
Jan 6, 2011·Applied Biochemistry and Biotechnology·Matthew B KerbyAnubhav Tripathi
Sep 5, 2007·Assay and Drug Development Technologies·Mingjie ZhouThomas L Fare
May 16, 2012·BMC Genomics·Mario Fasold, Hans Binder
May 22, 2007·BMC Biotechnology·William LonerganSuzanne D Vernon
Jun 29, 2007·Plant Methods·M Lia Molas, John Z Kiss
Oct 25, 2011·The Journal of Molecular Diagnostics : JMD·Weihua TangMargaret L Gulley
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Dec 18, 2009·Expert Review of Molecular Diagnostics·Federico A Monzon, Catherine I Dumur
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Feb 4, 2010·Archives of Pathology & Laboratory Medicine·Federico A Monzon, Tracie J Koen

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