Nucleic Acids Research, 2005, Vol. 33, No. 19 e166 doi:10.1093/nar/gni156 Position of the fluorescent label is a crucial factor determining signal intensity in microarray hybridizations Lei Zhang, Thomas Hurek and Barbara Reinhold-Hurek* Laboratory of General Microbiology, Center of Applied Gene Sensoric (CAG), University of Bremen, PO Box 33 04 40, D-28334 Bremen, Germany Received June 4, 2005; Revised August 8, 2005; Accepted September 21, 2005 ABSTRACT A key issue in applications of short oligonucleotide- based microarrays is how to design specific probes with high sensitivity. Some details of the factors affecting microarray hybridization remain unclear, hampering a reliable quantification of target nucleic acids. We have evaluated the effect of the position of the fluorescent label [position of label (POL)] relative to the probe-target duplex on the signal output of oligonucleotide microarrays. End-labelled single- stranded DNA targets of different lengths were used for hybridization with perfect-match oligonucleotide probe sets targeting different positions of the same molecule. Hybridization results illustrated that probes targeting the labelled terminus of the target showed significantly higher signals than probes targeting other regions. This effect was independent of the target gene, the fluorophore and the slide surface chemistry. Comparison of microarray signal patterns of fluorescently end-labelled, fluorescently internally random-labelled and radioactively end-labelled target-DNAs with the same set of oligonucleotide probes identified POL as a critical factor affecting signal intensity rather than binding efficiency. Our observations define a novel determinant for large differences of signal intensities. Application of the POL effect may contribute to better probe design and data interpretation in microarray applications. INTRODUCTION Increasingly, high-density oligonucleotide microarrays have been exploited for all ﬁelds of biological research on a large scale (1,2). Therefore, it is compulsory to obtain high speciﬁcity of oligonucleotide probes and high sensitivity of microarray hybridizations (1), especially for the application in the ﬁeld of single-nucleotide polymorphism (SNP) analysis and diagnostics. However, several key issues of this techno- logy such as the design of optimal oligonucleotide probes and their actual versus their predicted hybridization behaviour still have to be resolved (3). All microarray-based techniques for the analysis of DNA variation rest on the hybridization of complementary strands of DNA (4,5). The interpretation of microarray-based experiments relies on the signal intensity derived from labelled samples hybridized to the array. How- ever, many variables contribute to the level of the signal detec- ted. It has been often observed that some surface-bound probes targeting certain regions show low or even false negative signals, and even carefully designed oligonucleotide probes often display two orders of magnitude difference in signal output (6). Attempts to explain this behaviour include e.g. signal-suppressing parameters such as secondary structure of the target molecules, or steric hindrance of duplex forma- tion, selectively acting on different probe binding sites (4). Less attention appears to have been paid to the relative posi- tion of label (POL) with respect to the duplex formed. In the course of developing a diagnostic microarray for nitrogenase gene diagnostics (nifH phylochip), we compared in this study hybridization patterns of multiple surface-bound, short oligonucleotide probes targeting different regions of the target-DNAs. The results suggest that POL is a crucial factor determining signal intensity. Our observations provide an important insight into factors affecting microarray perform- ance and deﬁne a novel determinant for large differences of signal intensities. Application of the POL effect will contribute to an improved probe design and improved data interpretation in microarray applications. MATERIALS AND METHODS End-labelled targets The target sequences were fragments of the nifH gene encoding the nitrogenase iron protein of the nitrogen-ﬁxing *To whom correspondence should be addressed. Tel +49 421 218 2370; Fax: +49 421 218 9058; Email: breinhold@uni-bremen.de The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. 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