Development of quantitative multiplex RNA in situ hybridization

描述（由申请人提供）：正常和病变组织中细胞基因表达的可视化，结合组织学和病理分析，是理解生物基因功能及其对疾病过程贡献的关键一步。免疫组织化学（IHC）使用针对差异表达基因产物（或生物标志物）的特异性抗体，最能代表这一学科。代替分析蛋白质，可以通过原位杂交（ISH）检测mRNA转录物，原则上在以下方面提供了更大的灵活性：(1)探针合成（作为RNA转录物或合成寡核苷酸）；(2)通过探测同一转录物的不同片段来控制特异性；(3)检测非编码RNA和分析mRNA同种异构体。然而，传统的RNA ISH方案很难标准化，因为在ISH过程中，组织样品的制备方法和储存会影响RNA的完整性和检测，因为在组织切片的扩散过程中会丢失碎片RNA。我们重新分析了ISH过程的关键组成部分，包括RNA稳定性，RNA固定，探针设计和杂交，信号放大和检测，并建立了一个研究计划来纠正它的弱点。我们的发展已经产生了一种强大的方案，通过磷酸酰胺键形成将miRNA 5'-单磷酸与蛋白质氨基酸侧链交联，从而保留和检测组织切片中的miRNA。在这里，我们证明mRNA及其片段的ISH必须通过类似的方法来解决，以增加组织切片中的保留。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成为一种迅速超越传统免疫组化的技术。提供可靠的试剂和概念证明的演示将对癌症研究中的分子诊断和生物标志物的发展产生深远的影响。其他需要基于组织的诊断的医学领域，如与慢性病毒感染或移植有关的各种器官纤维化，也将受益于可靠的RNA ISH。作为原理证明，我们将利用RNA FISH监测肉瘤基因组计划中出现的诊断和预后价值转录本，通过更准确地分类小蓝圆细胞肿瘤（sbrct）和梭形细胞肿瘤（sct），应用该方法来提高肉瘤诊断。这些研究将与Samuel Singer领导的MSKCC肉瘤疾病管理团队和软组织肉瘤PO1研究者密切合作进行。总之，我们正在为一个可持续的发现和诊断RNA FISH平台建立化学基础，该平台将与传统的IHC竞争，因为它在RNA探针生产和多路复用方面具有灵活性，而且成本和时间都更低。它将结合经典组织学和先进的分子病理学，从而增加档案组织收藏与相关临床记录的价值。

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.