Development And Application Of Dual-ligation Probe For In Situ RNA Detection

Currently,RNA has become the beginning of most gene expression studies,so it is of great significance to accurately detect the expression abundance and spatial location of different RNAs.This project aims to develop a single-molecule RNA in situ detection method based on dual-ligation probe,so as to solve the problems of laborious process,low efficiency and limited types of detectable RNA in the single molecule RNA in situ detection based on reverse transcription.We first explored the development and application of dual-ligation probe for in situ mRNA detection,and established experimental schemes for single and multiple in situ mRNA detection.For single in situ mRNA detection,we discussed the strategy of dualligation probe design.And we found that for the different ligation ends in the same position on mRNA,the ligation efficiency of SplintR Ligase was as follows: CpT>TpA>ApT.And then we performed the multiple detection of four RNA biomarkers of HER2,ESR1,PGR and MKI67 on SKBR-3,MCF-7,MDA-MB-231 and T-47 D.The results showed ESR1 and PGR had the highest detection efficiency on T-47 D,with values of 6.22 and 25.27,respectively;while HER2 and MKI67 owned the greatest mean RCPs/cell on SKBR-3 and MDA-MB-231,which were 55.69 and 11.88 respectively.Besides,we found in the correlation verification experiment that the results obtained by our method showed a good linear correlation with the RNA sequencing data when using mRNA multiple detection(P<0.01).Based on the results we have obtained above,we then applied our method in lncRNA in situ detection.To that end,we conducted the multiple detection of MALAT1,NEAT1,CYTOR and H19 these four lncRNAs on MDA-MB-231,NCI-H1299 and T-47 D,which showed H19 and CYTOR were both distributed in the cytoplasm of MDAMB-231 and NCI-H1299.Moreover,we genotyped the three types of SNP of MALAT1_rs3200401 on A549,SW480,NCI-H460 and NCI-H1299,and we found both the amounts and intensity of non-target signals were extremely low,regardless of the SNP location at 3' or 5' end in dual-ligation probe,indicating good consistency existed with the SNP sequencing data.In addition,we used this method for multiple detection of miRNA,in which hsamiR-1244 and hsa-miR-3648 had relatively higher detection efficiency,and the means of RCPs/cell were 5.36 and 0.60,respectively.Finally,we accomplished the multiple detection of HER2,ESR1,PGR and MKI67 these four RNA biomarkers in formalinfixed paraffin-embedded breast cancer tissue samples,and three from eight tissue samples obtained ideal multiple detection results.In conclusion,thanks to the high efficiency,specificity,and sensitivity,our method is expected to be applied in basic biology,basic medicine and other fields of gene expression research at the RNA level,and it is more likely to develop into a novel clinical diagnostic technique.