Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA.
Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA.
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DOI：
10.1186/1472-6750-7-57
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发表时间：
2007-09-13
影响因子：
3.5
通讯作者：
Retief, Jacques
中科院分区：
文献类型：
--
作者：
Thompson, Karol L;Pine, P Scott;Rosenzweig, Barry A;Turpaz, Yaron;Retief, Jacques
微阵列数据的可解释性会受到样品质量的影响。为了系统地探索RNA质量如何影响微阵列分析性能，通过解冻冷冻组织或在一段时间内对新鲜组织进行离体孵育，生成了一组RNA完整性逐渐变化的大鼠肝脏RNA样本。组织在37°C下孵育数小时对RNA完整性影响不大，但确实诱导应激反应基因和免疫细胞标记物转录水平的变化。相反，组织解冻导致RNA完整性迅速丧失。被鉴定为对RNA降解最敏感的探针组往往位于其转录末端上游超过1000个核苷酸的位置，类似于用于评估Affymetrix GeneChip®阵列样品质量的对照探针组的定位。与未降解的肝脏RNA相比，RNA完整性小于或等于7的样品显示假阳性显著增加，在对样品完整性最敏感的探针组中，真阳性的检测减少，在计算机模拟的变化中为1.5倍、2倍和4倍。虽然具有3'偏位探针选择设计的微阵列可以耐受中等水平的RNA降解，但在本研究中，我们确定了一个阈值，超过该阈值，可以观察到特异性和敏感性下降，与平均靶长度密切相关。这些结果突出了用捕获样本质量重要方面的指标注释微阵列数据的价值。
The interpretability of microarray data can be affected by sample quality. To systematically explore how RNA quality affects microarray assay performance, a set of rat liver RNA samples with a progressive change in RNA integrity was generated by thawing frozen tissue or by ex vivo incubation of fresh tissue over a time course. Incubation of tissue at 37°C for several hours had little effect on RNA integrity, but did induce changes in the transcript levels of stress response genes and immune cell markers. In contrast, thawing of tissue led to a rapid loss of RNA integrity. Probe sets identified as most sensitive to RNA degradation tended to be located more than 1000 nucleotides upstream of their transcription termini, similar to the positioning of control probe sets used to assess sample quality on Affymetrix GeneChip® arrays. Samples with RNA integrity numbers less than or equal to 7 showed a significant increase in false positives relative to undegraded liver RNA and a reduction in the detection of true positives among probe sets most sensitive to sample integrity for in silico modeled changes of 1.5-, 2-, and 4-fold. Although moderate levels of RNA degradation are tolerated by microarrays with 3'-biased probe selection designs, in this study we identify a threshold beyond which decreased specificity and sensitivity can be observed that closely correlates with average target length. These results highlight the value of annotating microarray data with metrics that capture important aspects of sample quality.