Elsevier

Genomics

Volume 83, Issue 6, June 2004, Pages 1169-1175
Genomics

Short Communication
Evidence of systematic expressed sequence tag IMAGE clone cross-hybridization on cDNA microarrays

https://doi.org/10.1016/j.ygeno.2003.12.010Get rights and content

Abstract

We present evidence of a potentially serious source of error intrinsic to all spotted cDNA microarrays that use IMAGE clones of expressed sequence tags (ESTs). We found that a high proportion of these EST sequences contain 5′-end poly(dT) sequences that are remnants from the oligo(dT)-primed reverse transcription of polyadenylated mRNA templates used to generate EST cDNA for sequence clone libraries. Analysis of expression data from two single-dye cDNA microarray experiments showed that ESTs whose sequences contain repeats of consecutive 5′-end dT residues appeared to be strongly coexpressed, while expression data of all other sequences exhibited no such pattern. Our analysis suggests that expression data from sequences containing 5′ poly(dT) tracts are more likely to be due to systematic cross-hybridization of these poly(dT) tracts than to true mRNA coexpression. This indicates that existing data generated by cDNA microarrays containing IMAGE clone ESTs should be filtered to remove expression data containing significant 5′ poly(dT) tracts.

Section snippets

Results

We examined data from three cDNA microarray experiments: two experiments performed by separate investigators using commercially manufactured single-dye spotted cDNA arrays at the University of Pittsburgh (GF400, GeneFilters Microarrays; Research Genetics, Carlsbad, CA, USA; Peters et al., unpublished data, and Field et al., unpublished data) and one two-dye (Cy3/Cy5) experiment published on the publicly available Stanford Microarray Database Web site [6]. The first spotted cDNA microarray

Expressed sequence tags

ESTs are short (200–500 bp) sequences derived from directionally cloned plasmid cDNA libraries. Typically, total mRNA is isolated from cells in a particular type of tissue, stage of development, pathological state (e.g., normal versus tumor), or environmental/nutritional state (e.g., heat shock). The mRNA is reverse-transcribed using an oligo(dT) sequence primed with a restriction site. The resultant cDNA is then cloned into a plasmid vector, isolated, and one-pass sequenced, with the sequence

TZD-treated adipocytes experiment

The first spotted cDNA microarray experiment consisted of a time-sequenced sampling of differential mRNA expression from 3T3L1 cultured mouse adipocytes treated with the insulin-sensitizing agent TZD (Peters et al., unpublished data). Cells were harvested in 5 ml of Trizol (Invitrogen Corp., Carlsbad, CA, USA) and RNA was extracted according to the manufacturer's instructions. RNA integrity for each sample was confirmed on formaldehyde/formamide agarose gels prior to microarray analysis. cDNA

Acknowledgements

The authors thank Jay Kadane, Takis Benos, and Joe Ramsey for their helpful comments. This work was supported by NASA Grant NCC2-1227 and the Copeland Fund of the Pittsburgh Foundation Grant D200-0251.

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