Development of quantitative multiplex RNA in situ hybridization

定量多重RNA原位杂交的发展

• 批准号: 8616355
• 负责人: THOMAS TUSCHL
• 金额: $ 34.58万
• 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/8616355
• 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; public health relevance; 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.

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），它使用特异性差异表达的基因产物（或生物标志物）的抗体最好地代表了这一学科。代替分析蛋白质，可以通过原位杂交（ISH）检测mRNA转录物，其原则上在以下方面提供更大的灵活性：（1）探针合成（作为RNA转录物或合成寡核苷酸），（2）通过探测相同转录物的不同片段来控制特异性，和（3）检测非编码RNA和分析mRNA同种型。然而，传统的RNA ISH方案难以标准化，因为组织样品制备方法和储存影响RNA完整性及其检测，这是由于在ISH过程中通过从组织切片扩散而损失片段化的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，以纠正其弱点。我们的发展已经导致了一个强大的协议，保留和检测组织切片中的miRNA，通过交联的miRNA 5 '-单磷酸蛋白质氨基酸侧链通过氨基磷酸酯键形成。在这里，我们证明，ISH的mRNA及其片段必须通过类似的方法来解决，以增加保留在组织切片。在基因组和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，现在将RNA ISH定位为快速超越常规IHC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，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。 作为一个原则的证明，我们将应用该方法，以提高肉瘤的诊断，通过更准确的分类小蓝圆细胞肿瘤（SBRCTs）和梭形细胞肿瘤（SCT）利用RNA FISH监测转录的诊断和预后价值出现从肉瘤基因组计划。这些研究将与Samuel Singer领导的MSKCC的肉瘤疾病管理团队和软组织肉瘤PO 1研究者密切合作进行。 总之，我们正在为可持续的发现和诊断RNA FISH平台建立化学基础，该平台将与传统的IHC相媲美，因为它在RNA探针生产和多路复用方面具有灵活性，成本和时间更低。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关的档案组织收藏的价值。