dc.description.abstract | MicroRNAs (miRNAs) are a cluster of small, non-coding RNA molecules, generally 18-22 nucleotides in length, that play important roles in regulating gene expression by binding to the target messenger RNAs (mRNAs) and inducing mRNAs degradation. Previous studies have shown several correlations between aberrant miRNAs expression and a variety of human diseases. Hence, typical miRNAs are considered as diagnostic and prognostic biomarkers. That is to say, the need of developing highly sensitive and specific detection methods is necessary. Current detecting methods include In Situ Hybridization (ISH), Real time PCR, northern blotting, and miRNA microarray profiling technology. Above these methods, only ISH provide miRNA information about both expression level and localization in a single cell level. However, the main challenge of using DNA oligonucleotides as detecting probe is that the results lack of specificity since the small size and the nature fragile characterization of the target miRNA. In this study, we applied an alternative DNA analogue, which contains site-specific neutral methyl phosphotriester internucleotide linkages (MPTE), shows improved the hybridization properties due to the reduction of electrostatic repulsion between the double strands MPTEDNA/RNA duplex. And the lipophilic character allow the probe transport through cell membrane easily. In Situ Hybridization methods were performed to visualize mimic exogenous miR-524-5p we transfected into HCT116 cell lines (human colon cancer cell lines) and the well-known oncomiR in colon cancer cell lines, endogenous miR-21, by 3’-digoxigenin (DIG) labelled MPTE modified probe. Through optimal design of the modification of MPTE, the results demonstrated improved hybridization efficiency while remaining detecting specificity. Based on the success of applying MPTE probe on detecting miRNA through ISH, we expected the potential ability of MPTE modified oligonucleotides developing into theoretic agent. | en_US |