Quantitative analysis of microRNAs in tissue microarrays by in situ hybridization.
Quantitative analysis of microRNAs in tissue microarrays by in situ hybridization.
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DOI：
10.2144/000113837
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发表时间：
2012-04
期刊：
影响因子：
2.7
通讯作者：
Rimm DL
中科院分区：
文献类型：
--
作者：
Hanna JA;Wimberly H;Kumar S;Slack F;Agarwal S;Rimm DL
微小RNA（miRNAs）已经成为癌症发病机制中的关键调节因子，其中它们可以充当癌基因或肿瘤抑制因子。大多数miRNA测量方法需要总RNA提取物，其缺乏关键的空间信息，并且对标准化提出了挑战。我们已经开发并验证了一种通过原位杂交（ISH）定量分析miRNA表达的方法，该方法允许直接评估肿瘤上皮细胞的miRNA表达。这种基于共定位的方法（称为qISH）利用DAPI和细胞角蛋白免疫荧光在肿瘤上皮中建立亚细胞区室，然后与miRNA ISH多重化，允许定量测量这些区室内的miRNA表达。我们使用这种方法在组织微阵列上评估473例乳腺癌标本中miR-21、miR-92 a、miR-34 a和miR-221的表达。我们发现miR-221水平在乳腺癌中具有预后性，这说明了高通量方法，并证实了miRNA可以成为癌症中有价值的生物标志物。此外，在应用这种方法时，我们发现在大的群体队列中很难辨别miRNAs和所提出的靶蛋白之间的反比关系。我们的方法展示了一种基于组织微阵列的大队列miRNA表达评估方法。
MicroRNAs (miRNAs) have emerged as key regulators in the pathogenesis of cancers where they can act as either oncogenes or tumor suppressors. Most miRNA measurement methods require total RNA extracts which lack critical spatial information and present challenges for standardization. We have developed and validated a method for the quantitative analysis of miRNA expression by in situ hybridization (ISH) allowing for the direct assessment of tumor epithelial expression of miRNAs. This co-localization based approach (called qISH) utilizes DAPI and cytokeratin immunofluorescence to establish subcellular compartments in the tumor epithelia, then multiplexed with the miRNA ISH, allows for quantitative measurement of miRNA expression within these compartments. We use this approach to assess miR-21, miR-92a, miR-34a, and miR-221 expression in 473 breast cancer specimens on tissue microarrays. We found that miR-221 levels are prognostic in breast cancer illustrating the high-throughput method and confirming that miRNAs can be valuable biomarkers in cancer. Furthermore, in applying this method we found that the inverse relationship between miRNAs and proposed target proteins is difficult to discern in large population cohorts. Our method demonstrates an approach for large cohort, tissue microarray-based assessment of miRNA expression.
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