Cross-species hybridisation of pig RNA to human nylon microarrays

Abstract

Background: The objective of this research was to investigate the reproducibility of cross-species microarray hybridisation. Comparisons between same- and cross-species hybridisations were also made. Nine hybridisations between a single pig skeletal muscle RNA sample and three human cDNA nylon microarrays were completed. Three replicate hybridisations of two different amounts of pig RNA, and of human skeletal muscle RNA were completed on three additional microarrays.

Results: Reproducibility of microarray hybridisations of pig cDNA to human microarrays was high, as determined by Spearman and Pearson correlation coefficients and a Kappa statistic. Variability among replicate hybridisations was similar for human and pig data, indicating the reproducibility of results were not compromised in cross-species hybridisations. The concordance between data generated from hybridisations using pig and human skeletal muscle RNA was high, further supporting the use of human microarrays for the analysis of gene expression in the pig. No systematic effect of stripping and re-using nylon microarrays was found, and variability across microarrays was minimal.

Conclusion: The majority of genes generated highly reproducible data in cross-species microarray hybridisations, although approximately 6% were identified as highly variable. Experimental designs that include at least three replicate hybridisations for each experimental treatment will enable the variability of individual genes to be considered appropriately. The use of cross-species microarray analysis looks promising. However, additional validation is needed to determine the specificity of cross-species hybridisations, and the validity of results.

Figure 1

Microarray image generated from the hybridisation of pig skeletal muscle total RNA to the GF211 human nylon cDNA microarray (ResGen) containing 4,324 human gene and EST sequences.

Figure 2

Scatterplots of data generated from two replicate hybridisations of pig or human skeletal muscle RNA to a human cDNA nylon microarray. Data are median normalized and log transformed results from two hybridisations of (A) 4 μg of human RNA, (B) 2 μg of pig RNA, (C) 4 μg of pig RNA, or (D) 6 μg of pig RNA.

Figure 3

Scatterplots of standard deviations calculated from three replicate hybridisations of pig or human skeletal muscle RNA to a human cDNA nylon microarray. Standard deviations and means for each gene were calculated from median normalized and log transformed results from three hybridisations of (A) 4 μg of human RNA, (B) 2 μg of pig RNA, (C) 4 μg of pig RNA, or (D) 6 μg of pig RNA.

Figure 4

Distribution of variable genes across expression categories. Genes were categorized according to their average median normalized log transformed intensity (undetectable 0; low, 0–1.3; medium, 1.3–3; or high, ≥ 3). Variable genes were identified as genes with a standard deviation greater than two times the average standard deviation of the human data. The total number of genes in each expression category, and the number of genes variable in pig, human, or both, are shown.

Figure 5

Comparison of gene expression profiles generated from hybridisations of pig and human skeletal muscle RNA with a human cDNA nylon microarray. Data shown are the average median normalized and log transformed intensity of three hybridisations of human and pig skeletal muscle RNA with the GF211 human nylon cDNA microarray (ResGen) containing 4,324 human gene and EST sequences.

Figure 6

Density plot of the distribution of standard deviations. Standard deviations were calculated from median normalized log transformed data of three replicates each of human, 4 μg RNA (black); pig, 2 μg (blue); pig, 4 μg (red); and pig, 6 μg (green) hybridisations.

Figure 7

Density plot of the distribution of standard deviations.(A) Standard deviations were calculated from median normalized log transformed data of three replicates each from hybridisations representing the third (blue), fourth (black), and fifth (red) use of the filters. (B) Standard deviations were calculated from median normalized log transformed data of three replicates each from hybridisations using Filter 4 (black), Filter 5 (blue), and Filter 6 (red).