Transcriptome analysis in non-model species: a new method for the analysis of heterologous hybridization on microarrays

Abstract

Background: Recent developments in high-throughput methods of analyzing transcriptomic profiles are promising for many areas of biology, including ecophysiology. However, although commercial microarrays are available for most common laboratory models, transcriptome analysis in non-traditional model species still remains a challenge. Indeed, the signal resulting from heterologous hybridization is low and difficult to interpret because of the weak complementarity between probe and target sequences, especially when no microarray dedicated to a genetically close species is available.

Results: We show here that transcriptome analysis in a species genetically distant from laboratory models is made possible by using MAXRS, a new method of analyzing heterologous hybridization on microarrays. This method takes advantage of the design of several commercial microarrays, with different probes targeting the same transcript. To illustrate and test this method, we analyzed the transcriptome of king penguin pectoralis muscle hybridized to Affymetrix chicken microarrays, two organisms separated by an evolutionary distance of approximately 100 million years. The differential gene expression observed between different physiological situations computed by MAXRS was confirmed by real-time PCR on 10 genes out of 11 tested.

Conclusions: MAXRS appears to be an appropriate method for gene expression analysis under heterologous hybridization conditions.

Figure 1

Comparison of the distribution of fluorescence in microarrays with homologous (C, chicken) or heterologous (NI1 to NI4, never-immersed penguins and SA1 to SA3, sea-acclimatized penguins) hybridizations. All samples were hybridized on Affymetrix GeneChip^® Chicken Genome Arrays. Data for the homologous hybridization (C) were downloaded from the Gene Expression Omnibus (GSM157808). A: Fluorescence intensity boxplot. B: Fluorescence intensity density plot.

Figure 2

For a given probe set, the same probe had the largest fluorescence intensity in the majority of arrays. This figure represents the distribution of the maximum rank sum of the probes in each probe set divided by the number of probes corresponding to this probe set. If, for a given probe set, the same probe had the highest fluorescence intensity in all 7 microarrays considered here, we expected that this maximum rank sum divided by the number of probes would equal 7.

Figure 3

Scatter plot comparing gene expression between penguins before (NI) and after sea acclimation (SA). Each point represents the mean expression level of a gene in NI and SA conditions. Black dots represent differentially expressed genes between both situations. Red symbols represent the differentially expressed genes tested by qPCR: red stars correspond to the validated genes and red crosses to non-validated ones.

Figure 4

Comparison of the gene expression differences assessed by our heterologous hybridization analysis and by qPCR. A: Expression fold changes of the 11 genes tested by quantitative PCR. These fold changes correspond to SA/NI for the genes up-regulated during the transition from terrestrial to marine life (represented above the x-axis), and to NI/SA for the down-regulated genes (represented below the x-axis). The white bars correspond to the fold changes assessed by microarray and the black bars to the fold changes assessed by quantitative PCR. B: Comparison of the fold changes assed by microarray and by qPCR. Pearson correlation coefficient = 0.68 (p-value = 0.02).