Development And Applications Of A Novel Multiplex In Situ RNA Profiling Method

Multiplex in situ RNA profiling offers a new opportunity for gene expression by providing the expression abundance and spatial information.In this work,we present a novel single-molecule RNA multiple in situ detection technology based on padlock probes,which is intended to solve the problems of cumbersome process,low efficiency of single-molecule RNA based on reverse transcription and limitation of spectrally-resolved fluorescence dyes available.We tested our method in different mRNA in situ detection and established experimental schemes for multiple in situ mRNA detection.For multiplex in situ RNA profiling,we discussed the strategy of a novel padlock probes design.There are three positions on the padlock probes for insertion of barcode DNA fragments,each position contains one barcode DNA fragment from a set of four different sequences.After three interrogation cycles,they can encode 64 genes.And then we performed multiple detection on breast cancer-related genes such as HER2,ESR1,PGR and MKI67 in three breast cancer cell lines,SK-BR-3,MDA-MB-231 and T-47 D.The results showed ESR1 and PGR had the highest detection efficiency on T-47D;while HER2 and MKI67 owned the greatest mean RCPs/slide on SK-BR-3 and MDA-MB-231.And results are consistent with those in the Human Protein Atlas database.Besides,we correlated our multiplexed in situ hybridization expression data in SK-BR-3 and T-47 D with TPM values shown in the Human Protein Atlas database and the expression data in MDA-MB-231 with FPKM measured by RNA-seq,which showed a good linear correlation with the RNA sequencing data when using mRNA multiple detection(P<0.01).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.Based on the results we have obtained above,we then applied this method to formalin-fixed paraffin-embedded breast cancer tissue samples and analyzed theexpression of 39 different mRNAs quantitatively in three different subtypes breast cancer tissue samples,and then achieved an desired effect.The detected mRNA showed different localization patterns and the amount of signal detected is also disparate.Numerous mRNA spots could be readily detected in tumor regions densely populated by tumor cells,but not in the adjacent stroma.This also showed that our method has good specificity.Finally,this method have been applied to profile gene expression in brain tissue sections and very promising results in identifying different cell types in their original resident niche were obtained.Firstly,probes were designed and tested for multiple housekeeping genes of ants to confirm the feasibility of our experimental scheme in the ant brain.Secondly,the results showed that the gene expression patterns were quite different in at different focal depths of one ant slices.This also proves their great potential in neuroscience applications.In summary,due to the high detection efficiency,specificity and wide applicability,our method is expected to be applied to the study of gene expression at the RNA level in basic biology and basic medicine and the platform is expected to become a new clinical diagnostic technique.