The T7-Primer Is a Source of Experimental Bias and Introduces Variability between Microarray Platforms

Eberwine(-like) amplification of mRNA adds distinct 6-10 bp nucleotide stretches to the 5' end of amplified RNA transcripts. Analysis of over six thousand microarrays reveals that probes containing motifs complementary to these stretches are associated with aberrantly high signals up to a hundr...

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Bibliographic Details
Published in:PloS one Vol. 3; no. 4; p. e1980
Main Authors: Kerkhoven, Ron M., Sie, Daoud, Nieuwland, Marja, Heimerikx, Mike, De Ronde, Jorma, Brugman, Wim, Velds, Arno
Format: Journal Article
Language:English
Published: United States Public Library of Science 23.04.2008
Public Library of Science (PLoS)
Subjects:
Aberration
Acids
Antisense RNA
Bacteriophage T7 - metabolism
Base Sequence
Bias
Binding Sites
Breast cancer
Datasets
DNA microarrays
DNA Primers - genetics
DNA Primers - metabolism
DNA probes
DNA, Intergenic - genetics
DNA-Directed RNA Polymerases - metabolism
Enzymes
Gene expression
Gene Expression Regulation
Gene sequencing
Genetics and Genomics/Bioinformatics
Genetics and Genomics/Gene Expression
Genomes
Humans
Hybridization
Messenger RNA
Molecular Biology/Bioinformatics
Molecular Sequence Data
Oligonucleotide Array Sequence Analysis
Operon - genetics
Platforms
Probes
Quality control
Ribonucleic acid
RNA
RNA, Messenger - genetics
RNA, Messenger - metabolism
RNA, Viral - genetics
Studies
Viral Proteins - metabolism
Netherlands
ISSN:1932-6203, 1932-6203
Online Access:Get full text
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Description
Summary:Eberwine(-like) amplification of mRNA adds distinct 6-10 bp nucleotide stretches to the 5' end of amplified RNA transcripts. Analysis of over six thousand microarrays reveals that probes containing motifs complementary to these stretches are associated with aberrantly high signals up to a hundred fold the signal observed in unaffected probes. This is not observed when total RNA is used as target source. Different T7 primer sequences are used in different laboratories and platforms and consequently different T7 primer bias is observed in different datasets. This will hamper efforts to compare data sets across platforms.
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Conceived and designed the experiments: RK. Performed the experiments: DS MN MH WB. Analyzed the data: DS Jd AV. Contributed reagents/materials/analysis tools: DS MN MH Jd WB AV. Wrote the paper: RK DS Jd AV.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0001980