A Comprehensive Hybridization Model Allows Whole HERV Transcriptome Profiling Using High Density Microarray. Homo sapiens

Human endogenous retroviruses (HERVs) have received much attention for their implications in the etiology of many human diseases and their profound effect on evolution. Notably, recent studies have highlighted associations between HERVs expression and cancers [1], autoimmunity [2] and neurological [3] conditions. Their repetitive nature makes their study particularly challenging, where expression studies have largely focused on individual loci [4] or general trends within families [5, 6, 7]. To refine our understanding of HERVs activity, we introduce here a new microarray, HERV-V3, that offers an almost complete coverage of HERVs and their ancestors (mammalian apparent LTR-retrotransposons, MALRs) at the locus level. This was made possible by the careful detection and annotation of genomic HERVs/MALRs sequences as well as the development of a new hybridization model, allowing the optimization of probe performances and the control of cross-reactions. Although no gold standard in HERVs measurement exists to validate the array, HERV-V3 analytical performances were comparable to commercial Affymetrix arrays, and for a selection of tissue/pathological specific loci, the patterns of expression found on HERV-V3 were consistent with those reported in the literature. Overall design: HERV-V3 technical performances were evaluated on the MAQC samples, composed of two independent samples (A, Stratagene Universal RNA, and B, Ambion Human Brain RNA) from which two titration samples were generated (C and D, consisting of 3:1 and 1:3 ratios of A to B, respectively). Each sample was performed in technical triplicate. The biological validation was performed on three different tissues (colon, placenta and prostate) and two primary human cell lines (OSCAR and EBJ14).

人类内源性逆转录病毒（human endogenous retroviruses, HERVs）因与多种人类疾病的病因学关联以及对进化的深远影响而受到广泛关注。值得注意的是，近期研究揭示了HERVs表达与癌症[1]、自身免疫性疾病[2]及神经系统病症[3]之间的关联。
这类序列的重复性特征使其研究极具挑战性，此前的表达研究大多聚焦于单个基因座[4]或家族内的整体趋势[5,6,7]。
为深化对HERVs活性的认知，本研究推出一款新型微阵列芯片HERV-V3，该芯片可在基因座水平上几乎完整覆盖HERVs及其祖先序列——哺乳动物显性LTR逆转录转座子（mammalian apparent LTR-retrotransposons, MALRs）。这一成果得益于对基因组中HERVs/MALRs序列的精准检测与注释，以及全新杂交模型的开发，该模型可优化探针性能并控制交叉反应。
尽管目前尚无HERVs定量检测的金标准可用于验证该芯片，但HERV-V3的分析性能可与商用Affymetrix芯片相媲美；针对部分组织/病理特异性基因座，HERV-V3检测得到的表达模式与文献报道结果一致。
整体实验设计：采用微阵列质量控制（MicroArray Quality Control, MAQC）样本评估HERV-V3的技术性能，该样本集包含两份独立样本（样本A：Stratagene通用RNA，样本B：Ambion人脑RNA），并由此制备两份梯度稀释样本（样本C与D，分别为A与B按3:1及1:3的比例混合所得）。每份样本均设置3次技术重复。生物学验证则针对3种不同组织（结肠、胎盘及前列腺）与2种原代人细胞系（OSCAR及EBJ14）开展。