Development of quantitative multiplex RNA in situ hybridization

定量多重RNA原位杂交的发展

• 批准号: 8087185
• 负责人: THOMAS TUSCHL
• 金额: $ 36.99万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8087185
• 关键词: Address; Affect; Amino Acids; Antibodies; Area; Automation; Biological; Biological Markers; Calibration; Carbodiimides; Cells; Chemicals; Chronic; Classification; Clinical; Collaborations; Detection; Development; Diagnosis; Diagnostic; Diffusion; Discipline; Disease; Disease Management; Fibrosis; Fluorescence Microscopy; Fluorescent in Situ Hybridization; Foundations; Functional RNA; Gene Expression; Genes; Genome; Haptens; Histology; Imagery; Imidazole; Immunohistochemistry; In Situ Hybridization; Label; Link; Measures; Medical; Memorial Sloan-Kettering Cancer Center; Messenger RNA; Methods; MicroRNAs; Molecular; Monitor; Oligonucleotide Probes; Oligonucleotides; Organ; Polystyrenes; Positioning Attribute; Preparation; Process; Production; Protein Isoforms; Proteins; Protocols documentation; RNA; RNA Probes; RNA Sequences; RNA Stability; RNA analysis; Reagent; Records; Research; Research Personnel; Sampling; Scanning; Side; Signal Transduction; Slide; Specificity; Spindle Cell Neoplasm; Technology; Testing; Time; Tissue Banks; Tissue Sample; Tissues; Transcript; Transplantation; Virus Diseases; antibody conjugate; anticancer research; base; cell type; cost; crosslink; design; flexibility; gene function; genome sequencing; image processing; improved; molecular pathology; novel strategies; phosphoramidate; prognostic; programs; sample fixation; sarcoma; soft tissue; tissue processing

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.

描述（由申请人提供）：正常和患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，是理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) 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。可以通过原位杂交 (ISH) 来检测 mRNA 转录本，而不是分析蛋白质，原则上，原位杂交在以下方面提供了更大的灵活性：(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 协议很难标准化，因为组织样本制备方法和储存会影响 RNA 的完整性及其检测，因为在 ISH 过程中会因组织切片的扩散而导致片段化 RNA 的损失。 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其弱点。我们的开发已经形成了一个强大的方案，通过通过氨基磷酸酯键形成将 miRNA 5'-单磷酸与蛋白质氨基酸侧链交联，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、组织处理自动化以及荧光显微镜扫描和图像处理等领域的最新进展，现在使 RNA ISH 成为一种迅速超越传统 IHC 的技术。提供可靠的试剂和概念验证的演示将对癌症研究中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疗领域，例如与慢性病毒感染或移植相关的各种器官的纤维化，也将受益于可靠的 RNA ISH。 作为原理证明，我们将利用 RNA FISH 监测肉瘤基因组项目中具有诊断和预后价值的转录本，通过对小蓝色圆形细胞肿瘤 (SBRCT) 和梭形细胞肿瘤 (SCT) 进行更准确的分类，应用该方法来改进肉瘤诊断。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 研究人员密切合作进行。 总之，我们正在为可持续发现和诊断 RNA FISH 平台奠定化学基础，该平台由于其在 RNA 探针生产和多重分析方面的灵活性，能够以更低的成本和时间与传统 IHC 相媲美。它将结合经典组织学和先进的分子病理学，从而提高档案组织收藏与相关临床记录的价值。 公共健康相关性：该项目描述了更好地测量正常和患病组织中 RNA 水平基因表达的新方法，并将经典组织学和先进分子病理学结合起来。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，旨在通过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更准确的分类来促进肉瘤诊断。