During the last decade, the use of DNA microarrays has become a key tool in molecular biology. This technology is commonly used for physiological and medical studies to generate snapshots of gene expression patterns in tissues of organisms exposed to different environmental conditions, allowing us to infer regulatory pathways involved in cellular responses to these conditions. The increased prevalence of microarray technology has benefited from the emergence of easily available commercial arrays. However, commercial microarrays target a limited number of species. Moreover, for many non-traditional model organisms, the insufficient amount of sequence data prevents the development of dedicated microarrays. Therefore, a few studies have investigated the use of heterologous array hybridization, [i.e. hybridization on arrays designed for a particular species (hereafter called the reference species) to explore modifications of gene expression patterns of another species (hereafter called the studied species)] and highlighted the difficulties inherent to this approach.
Heterologous hybridization is usually considered a non-standard utilization of microarrays . Indeed, it raises a number of difficulties, essentially due to the sequence divergence between the reference and the studied species . A major consequence of heterologous hybridization is a global reduction of hybridization fluorescence signal ( and references therein). This reduction artificially decreases the number of differentially expressed genes detected by standard statistical tests, leading to a misrepresentation of the variation in transcriptomic profiles ( and references therein). Another issue of heterologous hybridization is cross-hybridization . Indeed, microarrays are designed so that each probe is specific to one transcript sequence in the dedicated species. However, this specificity is not guaranteed when transcripts from another species are hybridized onto the array. On the other hand, the use of heterologous hybridization does not amplify the problem of differentiating paralog expression levels compared to the use of the dedicated platform species.
For all these reasons, the use of heterologous hybridization should be preceded by a careful choice of the type of microarray to use and followed by an appropriate analysis of the results.
To choose the most appropriate microarray to use, one has to select the model organism with the lowest sequence divergence from the studied species . Due to the lack of sufficient sequence data for all studied species, expression profiling results are the most robust when using microarrays dedicated to the reference species with the smallest phylogenetic distance from the studied species .
Once the reference species is chosen, one has to choose the best type of probe to use: either short oligonucleotide probes, such as those on Affymetrix GeneChips®, or longer probes, such as long oligomers or even full-length cDNAs. Microarrays with long probes might be less sensitive to sequence mismatches and thus facilitate heterologous hybridization [1–3, 6]. However, most arrays with long probes contain only one probe per transcript. It can be advantageous to use arrays with several short probes targeting the same transcript: the sequence of some probes may be more similar to the orthologous sequence in the species of interest than others. Therefore, one can consider only those the probes that recognize conserved areas of genes between reference and studied species [3, 7, 8]. These specific probes can be determined from sequence comparison [3, 8] or experimentally after hybridizing genomic DNA to the microarray . However, the lack of sufficient sequence data in many species prevents the determination by sequence comparison, and the hybridization of genomic DNA raises the problem of setting the threshold of fluorescence to accept or reject the information provided by a probe .
In the present study, we were interested in gene expression changes in the pectoralis muscle of juvenile king penguins at a key step of their development, during the transition from terrestrial to marine life. Strictly terrestrial during their first year after hatching, king penguin chicks must then depart to sea to become self-sufficient, and pectoralis muscle is largely involved in penguin adaptation to the marine environment . We choose the chicken as our reference species, as this is the closest model species for which microarrays are available. Chicken and king penguin are separated by approximately 100 millions years of phylogenetic divergence . We decided to use Affymetrix GeneChip® Chicken Genome Arrays because they present on average 11 different probe pairs per probe set (i.e., a set of perfect-match and mismatch probes targeting one given transcript), which should increase the probability that at least one probe will hybridize with the heterologous transcript. We then developed a new method (MAXRS, for maximum rank sum) to analyze heterologous hybridization transcriptomic profiles. This method takes advantage of the design of Affymetrix microarrays with different probes targeting the same transcript. Statistical analyses were then conducted to identify differentially expressed genes in the pectoralis muscle between never-immersed and sea-acclimated penguins. Finally, we confirmed by quantitative PCR the expression profiles of 10 up- or down-regulated genes exhibiting a wide range of fold changes, out of 11 tested. MAXRS therefore appears to be an appropriate method of gene expression analysis under heterologous hybridization conditions and provides new perspectives in the application of microarray technology to ecological physiology studies.