Development Of RNA In Situ Detection Technology And Its Application In LncRNA Detection

RNA expression is highly heterogeneous.Accurate detection of RNA expression abundance and spatial location can help us understand the dynamic genes expression of cells and tissues at different times,and better understand the mechanism of action between body components.The aim of this study is to develop a novel single-molecule RNA in situ detection technique for the quantitative localization of target gene expression using dual-ligation probes,and to construct spatial gene expression profiles more efficiently.Based on the amplification of the padlock probe and rolling circle amplification,the main innovation of this research is to design new dual-ligation probes.By examining the design principle of the dual-ligation probes,the test compares the detection efficiency and applies it to the detection of different kinds of RNA.We have established a method for in situ detection of RNA based on rolling circle amplification using dual-ligation probes.Firstly,the ACTB housekeeping gene was used as a model to detect the reaction by analog-padlock probes.By optimizing reverse transcription,dual-ligation probes first-step ligation and splint-based circle-forming reaction system,the basic method detection of RNA based on dual-ligation probes was established.Comparing the method of dual-ligation probes and padlock probe,it is concluded that the dual-ligation probes are more efficient than the padlock probe.This method was then extended to the detection of other mRNAs to examine KRAS and breast cancerassociated genes in cell slides and histopathological sections,and to examine the specificity and robustness of the method.The method was then applied to the multiplex detection of cancer cell lncRNA and the simultaneous detection of mRNA and lncRNA,again demonstrating the robustness of the method.When the lncRNA SNP site is detected,the false positive rate can be reduced to less than 1% by changing the probe design,washing conditions,ligase and reaction system.In this paper,the dual-ligation probes and the padlock probe were compared several times to verify that the method is more efficient.At the same time,through the detection experiment of SNP locus,a higher single base recognition specificity has been achieved.This approach can be combined with a variety of in situ sequencing technologies to achieve in situ detection of single-cell,single-molecule,single-base RNA highly multiplex gene expression.Due to the consistency of in situ detection results with clinical information,this method is expected to become a new technology for basic research and clinical diagnosis.