Abstract
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.
Highlights
Amplification of messenger RNA is widely accepted as the method of choice to acquire sufficient copies of mRNA transcripts to enable microarray expression analysis of experimental samples [1,2]
The T7 primer used in amplification of mRNA for microarray applications is a potential source for bias in microarray data
The bias appears to be common to most microarray platforms that use amplification and mean intensities of one to nine percent of the probes increase aberrantly to levels up to a hundred fold the average level of unaffected probes
Summary
Amplification of messenger RNA is widely accepted as the method of choice to acquire sufficient copies of mRNA transcripts to enable microarray expression analysis of experimental samples [1,2]. The method is robust, very reproducible and it yields high quality antisense RNA material which can be labeled with fluorescent dyes and hybridized to sense oligonucleotide microarrays. Besides the mentioned discrepancies between total RNA and amplified RNA, additional variation in results is observed when the same amplified RNA sample is analyzed on different microarray platforms [5,6]. We assumed that the precise sequence of the T7 primer is important in this respect because the complete T7-primer is incorporated in the first strand cDNA generated by an RT enzyme upon annealing to the poly-A tail of mRNA extracted from samples
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