Comparison of cross-species and species-specific hybridizations

Abstract Background: One of the approaches for conducting genomics research in organisms that do not yet have a proper microarray template is to profile their expression patterns by using cross-species hybridization (CSH). Several different studies using spotted microarray for CSH resulted with contradicting conclusions as to the ability of CSH to reflect biological processes. Results: We used a tomato spotted cDNA microarray to examine the ability of CSH to reflect species specific hybridization (SSH) data. Potato RNA was hybridized to spotted cDNA tomato and potato microarrays to generate heterologous and homologous hybridization data, respectively. The results revealed difficulties in obtaining transcriptomics data from CSH that reflected those obtained from SSH. Nevertheless, once the data was filtered for those corresponding to matching probe sets, by restricting proper cutoffs of probe homology, the CSH transcriptomics data better reflected those of the SSH, to an extent that was quantitated by identification of differentially regulated genes. Conclusions: This study enabled us to outline some considerations regarding evaluation of a microarray as candidate platform for CSH study, performance of CSH and proper data analysis that may allow CSH to reflect to some extent a biological process. Keywords: cross-species hybridization; heterologous hybridization To examine whether cross species hybridization (CSH) can produce data that reflects species specific hybridization (SSH) data, we used the same RNA samples for both CSH and SSH microarray experiments. RNA samples were extracted from nematode-infected and non-infected (control) potato plants, sampled at two time points (at 5 and at 10 days of nematode infection). These RNA samples were co-hybridized with a reference sample by CSH to a tomato microarray and by SSH to a potato microarray. The reference sample was pooled from all 8 samples (2 time points of infected or non-infected plants; 2 biological replicates were performed). The CSHs were designated as 'PT' for potato samples on tomato microarrays; the SSHs were designated as 'PP' for potato samples on potato microarrays.

【摘要 背景】对于尚未拥有合适微阵列模板的生物体，开展基因组学研究的方法之一是借助跨物种杂交（cross-species hybridization, CSH）来解析其基因表达模式。既往多项采用斑点微阵列开展跨物种杂交的研究，在跨物种杂交能否反映生物学过程这一问题上得出了相互矛盾的结论。

【结果】本研究使用番茄斑点cDNA微阵列，探究跨物种杂交能否反映种特异性杂交（species specific hybridization, SSH）数据。我们将马铃薯RNA分别与斑点cDNA番茄微阵列、马铃薯微阵列进行杂交，分别获得异源杂交与同源杂交数据。结果显示，从跨物种杂交实验中获取能够匹配种特异性杂交结果的转录组数据存在难度。不过，当通过设置合理的探针同源性阈值，对匹配探针集对应的数据集进行过滤后，跨物种杂交的转录组数据能够更好地匹配种特异性杂交的结果，该优化程度可通过差异调控基因的鉴定进行量化。

【结论】本研究明确了若干考量要点，涉及作为跨物种杂交研究候选平台的微阵列评估、跨物种杂交实验实施以及恰当的数据分析策略，可使跨物种杂交在一定程度上反映生物学过程。

【关键词】跨物种杂交；异源杂交

为验证跨物种杂交能否生成匹配种特异性杂交的数据，我们将同一批RNA样本分别用于跨物种杂交与种特异性杂交的微阵列实验。RNA样本提取自线虫感染及未感染（对照）的马铃薯植株，采样时间点为线虫感染后的第5天与第10天。我们将这些RNA样本与参照样本混合后，分别通过跨物种杂交与番茄微阵列结合、种特异性杂交与马铃薯微阵列结合的方式完成杂交。参照样本混合自全部8份样本（感染/未感染植株各2个时间点，每组设置2次生物学重复）。跨物种杂交实验被标记为"PT"，即番茄微阵列上的马铃薯样本；种特异性杂交实验被标记为"PP"，即马铃薯微阵列上的马铃薯样本。