新型单分子RNA原位检测技术的开发及其在空间基因表达分析中的应用

RNA expression is a highly dynamic process that possesses extensive heterogeneity. Precise analysis of the expression level and spatial localization information of expressed RNA molecules can help us to better understand the physiological and pathological status of cells and tissues. This project aims to develop a novel single molecule RNA in situ detection method that is capable of achieving more efficient spatial gene expression analysis at single cell, single molecule and even single nucleotide level. Based on our previous exprience of developing single molecule RNA in situ detection and in situ sequencing for gene expression analysis, we will further invesigate and improve our newly developed dual-ligation probe assay based single molecule RNA in situ detection technique. By using the development of in situ detection of known gene expression and mutations in cultured cell and tissue samples as model assays, we will first establish a novel single molecule RNA in situ detection technique. Thereafter, by combining in situ sequencing technology, we will be able to develop a highly multiplex in situ gene expression detection technique. Finally, upon applying the technique on clinical cancer tissues, we will then establish a method for spatial gene expression profiling. There is a great potential for this novel technique being used in the field of basic biology and medical research, as well as in applications of clinical diagnostics.

RNA分子的表达是一个高度动态的过程并且具有广泛的异质性，准确地检测RNA的表达的丰度和空间位置信息能够帮助我们更好地了解细胞和组织的生理以及病理状态。本项目旨在开发一种新型单分子RNA原位检测技术，在单细胞、单分子和单碱基的水平上实现更高效率的空间基因表达分析。依托申请者前期开发单分子RNA原位检测和原位测序技术的经验，我们将对本课题组新近开发的双连接探针法单分子RNA原位检测技术进一步考察和改进，以在细胞和组织中对已知的基因表达和基因突变进行检测为模型，初步建立新型单分子RNA原位检测技术。接着结合原位测序技术，在单分子RNA水平上实现高度多重基因表达原位检测，并通过在临床肿瘤组织切片上的应用，总结和建立基于该技术的空间基因表达分析方法。本项目所建立的技术有望成为一种既可应用于生物学和医学基础研究，又可以用于临床诊断的分子生物学新技术。

分析基因表达的空间异质性有助于帮助我们更好的理解其在健康与疾病状态的细胞或者组织中所发挥的功能。基于此，本项目开发了可用于基因表达原位分析的新型的单分子RNA原位检测技术并在此基础上建立了基于改进型原位测序的靶向空间转录组学技术。基于双连接探针的新型单分子RNA原位检测技术，不仅能够实现对编码和非编码RNA的检测，还可以进行基因变异和选择性剪接等的原位检测分析，而改进型的原位测序技术则能够实现对100个以上的基因同时进行原位表达分析。利用这些技术，我们实现了在单细胞，甚至亚细胞水平的空间基因表达分析，以及进行基因表达的组织区域异质性的分析。我们所开发的新型RNA原位检测技术和改进型的原位测序技术可以作为空间多组学研究的技术手段，有望成为基础研究和临床诊断的新方法。