Aristaeus chip 1.0: an in situ synthesized oligonucleotide microarray for Ovis aries

Here we present a high-density in situ synthesized microarray for Ovis aries, named Aristaeus, designed by means of a pipeline of software instruments that, starting from non-annotated redundant EST sequences, selects oligonucleotides suitable for in situ generation on chip. The chip was tested by comparing the gene expression profiles of two sheep breeds with different phenotype, Sarda and Gentile di Puglia. We carried out microarray experiments on liver and udder tissues from lactating individuals and identified a relevant number of differentially expressed genes, all involved in metabolism pathways. The results are consistent with literature knowledge, while selected differential gene expressions have been confirmed by quantitative real-time polymerase chain reaction analyses. Tissue samples of liver were collected from 4 lactating individuals of two sheep (Ovis aries) breeds, Gentile di Puglia and Sarda. Biopsies of liver tissue were taken at second lactation stage (first record, stage 01: 6 days after lambing; second record, stage 02: 44 days after lambing) in both breeds. Tissues from liver were immersed in RNAlater (Sigma) immediately after biopsy and stored at -20°C. Samples were pooled by breed and then reverse labeled (cy5 and cy3), resulting in four raw data sets.

本研究构建了一款针对家绵羊（Ovis aries）的高密度原位合成微阵列（in situ synthesized microarray），命名为Aristaeus。该芯片依托一套软件工具流程设计而成，从未经注释的冗余表达序列标签（Expressed Sequence Tag, EST）序列中筛选出适用于芯片原位合成的寡核苷酸（oligonucleotide）。本研究通过对比两个表型存在差异的绵羊品种——撒尔达绵羊（Sarda）与真蒂莱迪普利亚绵羊（Gentile di Puglia）的基因表达谱，对该芯片进行了性能验证。我们对泌乳期个体的肝脏与乳腺组织开展了微阵列实验，鉴定出数量可观的差异表达基因，且所有差异基因均参与代谢通路。研究结果与已有文献报道相符，部分筛选出的差异表达基因已通过实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）得到验证。本研究从真蒂莱迪普利亚绵羊与撒尔达绵羊两个品种的4头泌乳个体中采集肝脏组织样本，两个品种的肝脏活检均于泌乳第二阶段开展：首次采样记为阶段01，即产羔后6天；第二次采样记为阶段02，即产羔后44天。肝脏组织活检完成后，立即将样本浸没于RNAlater（Sigma）中，并于-20℃条件下保存。按品种对样本进行混合后，进行反向标记（Cy5与Cy3），最终得到四组原始数据集。