Development of quantitative multiplex RNA in situ hybridization

描述(申请人提供)：正常和疾病组织中细胞基因表达的可视化，结合组织学和病理学分析，是了解生物基因功能及其对疾病过程的贡献的关键一步。免疫组织化学(IHC)是这一学科的最佳代表，它使用针对差异表达基因产物(或生物标记物)的特异性抗体。可以通过原位杂交(ISH)来检测mRNA转录本，而不是分析蛋白质，这在原则上提供了更多的灵活性：(1)探针合成(作为RNA转录本或合成寡核苷酸)，(2)通过探测同一转录本的不同片段来控制特异性，以及(3)检测非编码RNA和分析mRNA亚型。然而，传统的RNA ISH方法很难标准化，因为在ISH过程中，组织标本的制备方法和存储方法会影响RNA的完整性及其检测，因为在ISH过程中，组织切片中的碎片RNA会因扩散而丢失。我们重新分析了ISH过程中的关键环节，包括RNA的稳定性、RNA的固定、探针的设计和杂交、信号的扩增和检测，并制定了纠正其弱点的研究计划。我们的发展已经形成了一种可靠的协议，通过形成磷酰胺键将miRNA5‘-单磷酸与蛋白质氨基酸侧链交联，从而在组织切片中保留和检测miRNAs。在这里，我们证明了mRNA及其片段的ISH必须通过类似的方法来解决，以增加组织切片的保留率。在基因组和RNA测序、寡核苷酸探针设计、组织处理自动化、荧光显微镜扫描和图像处理等领域的最新进展，使RNA ISH成为一种迅速超过传统IHC的技术。提供可靠的试剂和概念验证的示范将对癌症研究中分子诊断学和生物标志物的发展产生深远的影响。其他需要组织诊断的医疗领域，如与慢性病毒感染或移植有关的各种器官的纤维化，也将受益于可靠的RNA ISH。作为原则的证明，我们将应用该方法通过更准确地对小蓝色圆形细胞肿瘤(SBRCT)和梭形细胞肿瘤(SCT)进行分类，利用RNA FISH来监测来自肉瘤基因组计划的具有诊断和预后价值的转录本，从而改进肉瘤诊断。这些研究将与肉瘤疾病管理团队和由塞缪尔·辛格领导的MSKCC的软组织肉瘤PO1研究员密切合作进行。总而言之，我们正在为可持续的发现和诊断RNA FISH平台建立化学基础，由于其在RNA探针生产和多路复用方面的灵活性，该平台将与传统的IHC相竞争，从而降低成本和时间。它将结合经典组织学和先进的分子病理学，从而增加与临床记录相联系的档案组织收藏的价值。 与公共卫生相关：该项目描述了更好地在正常和疾病组织的RNA水平上测量基因表达的新方法，并结合了经典组织学和高级分子病理学。将与纪念斯隆·凯特琳癌症中心的肉瘤疾病管理团队进行合作研究，目的是通过更准确地对不同类型的小蓝色圆形细胞肿瘤和梭形细胞肿瘤的基因表达进行分类，促进肉瘤诊断。

DESCRIPTION (provided by applicant): The visualization of cellular gene expression in normal and diseased tissues, combined with histological and pathological analysis, is a critical step towards understanding biological gene function and its contribution to disease processes. Immunohistochemistry (IHC), which uses antibodies specific to differentially expressed gene products (or biomarkers) best represents this discipline. Instead of analyzing proteins, mRNA transcripts can be detected by in situ hybridization (ISH), which in principle offers more flexibility in (1) probe synthesis (either as RNA transcript or synthetic oligonucleotide), (2) control of specificity by probing different segments of the same transcript, and (3) detection of non-coding RNAs and analysis of mRNA isoforms. However, conventional RNA ISH protocols are difficult to standardize because tissue sample preparation methods and storage affect RNA integrity and its detection due to loss of fragmented RNA by diffusion from tissue sections during the process of ISH. We have re-analyzed the critical components of the ISH process including RNA stability, RNA fixation, probe design and hybridization, signal amplification and detection, and established a research program to correct its weaknesses. Our developments already resulted in a robust protocol to retain and detect miRNAs in tissue sections by crosslinking the miRNA 5'-monophosphate to protein amino acid side chains via phosphoramidate bond formation. Here, we demonstrate that ISH of mRNA and its fragments has to be addressed by similar approaches to increase retention in tissue sections. Recent advances in such fields as genome and RNA sequencing, oligonucleotide probe design, automation of tissue processing, and fluorescence microscopy scanning and image processing, now position RNA ISH as a technology to rapidly surpass conventional IHC. Providing reliable reagents and demonstrations of proof of concepts will have a profound impact on the development of molecular diagnostics and biomarkers in cancer research. Other medical areas with a need for tissue-based diagnostics, such as fibrosis of various organs linked to chronic viral infection or transplantation would also benefit from reliable RNA ISH. As a proof of principle, we will apply the approach to improve sarcoma diagnosis through more accurate classification of small blue round cell tumors (SBRCTs) and spindle cell tumors (SCTs) utilizing RNA FISH for monitoring transcripts of diagnostic and prognostic value emerging from the sarcoma genome project. These studies will be carried out in close collaboration with the Sarcoma Disease Management Team and the Soft Tissue Sarcoma PO1 Investigators at MSKCC led by Samuel Singer. In summary, we are building the chemical foundation for a sustainable discovery and diagnostic RNA FISH platform that will rival conventional IHC due to its flexibility in RNA probe production and multiplexing at reduced cost and time. It will unite classical histology and advanced molecular pathology, thereby increasing the value of archival tissue collections with linked clinical records. PUBLIC HEALTH RELEVANCE: This project describes new approaches to better measure gene expression at the RNA level in normal and diseased tissues and unites classical histology and advanced molecular pathology. Collaborative studies will be conducted with the Sarcoma Disease Management Team at Memorial Sloan Kettering Cancer Center with the aim to facilitate sarcoma diagnosis through more accurate classification of gene expression in different types of small blue round cell tumors and spindle cell tumors.