Multiplex In Situ Profiling Of RNA Alternative Splicing In Hematopoietic Stem Cells

In situ detection of gene expression is able to provide its spatial location information at the cellular and tissue levels,and has attracted wide attention and applications.However,the same gene may produce multiple spliceosomes with different spatio-temporal specific expressions through selective splicing,and finally different spliceosomes will be translated into proteins and execute their biological functions.Therefore,it is of great biological significance to explore the spatial expression of gene selective splicing.The purpose of this study is to develop a novel RNA multiple in situ detection technique for the study of highly cell-specific gene selective splicing in hematopoietic stem cells.First,we used the leukemia cell line K562 that can differentiate into mature blood cells(Erythroblasts,EBs)and megakaryocyte(Megakaryocytes,MKs)as the foundation of the research model.We evaluated the efficiency of K562 differentiation by flow cytometry and used RNA in situ detection technology to localize specific expressed genes at the cellular level,and compared that with the transcriptome sequencing(RNA-seq)value of the proceeds of the gene expression,confirming the feasibility of the method for testing of gene expression.Secondly,based on the RNA-seq data analysis of K562,EBs and MKs,we selected alternative splicing events for in situ RNA detection experiments,and further optimized them for multiplex in situ RNA detection.Combined with the results of sequencing analysis,12 Circular RNA(Circular RNA,circRNA)were selected for detection on K562 and EBs cell lines.This method was used to detect the selective splicing events,and the results of the selective splicing events were analyzed to successfully classify the two cell lines.Finally,we applied the optimal conditions to mice bone marrow biopsy.The mice bone marrow microenvironment was first divided into 17 different cell types,47 different kinds of specific genes and 29 different circRNA were selected to be detected by RNA multiple in situ detection,thus,the gene expression map of bonemarrow microenvironment was analyzed through in-situ profiling,exploring the possible relationship between the genes of bone marrow microenvironment in mice.In summary,we have successfully detected specific genes and selective splicing events of different cell types by using above-mentioned method,successfully conducted typing at the level of hematopoietic stem cells and observed the spatial distribution at the level of single cell in bone marrow slices of mice.Because the typing of hematopoietic stem cells is quite complex,this method has high specificity and sensitivity,it is expected to be applied in basic research of stem cells and in-situ detection of cancer RNA based on selective splicing,which has the potential as a new clinical diagnostic method.