Methods For Detection Of MicroRNA And M~6A And The Application In Clinical Specimens

Mature miRNAs are a class of endogenous,single-stranded,non-protein-coding,small RNAs.As post-transcriptional regulators,miRNAs are widely exprssed in plants,animals and bacteria.miRNAs are not only involved in biological processes such as early development,cell differentiation,proliferation,hematopoietic function,immune response,but also closely related to disease and tumor development and prognosis.miRNA acts as an important biomarker,so detection of miRNA has great significance for the further understanding of molecular mechanisms,the diagnosis and treatment of diseases and the design of anti-cancer drugs.m6A is another important dynamic reversible epigenetic modification and plays a key role in the post-transcriptional regulation of RNA splicing,translation,stability,transportation and location.With the methylation and demethylation mechanisms of m6A revealed,it becomes clearly that the specific methylation loci and different methylation level of m6A have unique biological functions.Therefore,the detection of m6A becomes a great important part in epigenetics research.In this thesis,we summary the existing methods for detection of miRNA and m6A,moreover,some new technologies for analysis of miRNA and m6A have been developed and applied in clinical specimens.The main work in this thesis includes the following four parts:1.A method for Mutiplexed detection of miRNAs was developed based on conformational switch of molecular beacons(MB)and exonuclease.Molecular beacon can complementary hybridize with target miRNA and then the haripin structure was opend,exonuclease I was used to recognise and degraded ssDNA overhang of unfold-MB probe in the 3'-5 'direction,resulting in the 3' terminal quencher cleaved from the MB probe,and the fluorescence turned on.The intensity of fluorescence increased with the increasing concentration of miRNA,which could be used to quantify miRNA.More specially,by desiging different MB probes and modified them with multiple fluorophore-quencher pairs,our method had been used for multiplexed miRNA detection.Moreover,we applied this strategy for miR-21 assay in hepatocellular carcinoma(HCC)tissues.2.Visual detection of miRNA was achieved by coupling exonucleation reaction with dendritic rolling circle amplification.The exonucleation reaction was used to generate DNA:miRNA hybrid,then RNase H degraded miRNA,the remaining ssDNA fragement acted as template for ligation of padlock probe and primered the isothermal dendritic rolling circle amplification(DRCA).The DRCA products contained massive G-quadruplexs,it could bind with hemin to form peroxidase,which catalysed oxidation of ABTS2-to the colored product ABTS",and the LOD was lfM.Here,visible detection of miRNA in cells was realized by using this method,moreover,more platforms were provided for point-of-care testing(POCT).3.We developed an isothermal and ultrasensitive method for miRNA detection based on a target-promoted ligation and hyper-branched rolling circle amplification(HRCA).The miRNA was used to ligate the padlock probe in the prescence of SplintR ligase,and then triggered the HRCA.The HRCA products contained massive dsDNA and ssDNA,which could be quantified by SYBR Green(SG)I directly.This method had excellent sensitivity and specificity,and was applied to analyze the expression level of miR-21 in HCC tissues.4.A method based on single base extension reaction was reported to detect m6A.m6A hindered the DNA replication and RNA reserve by using Bst DNA polymerase.Therefore,we could distinguish methylation from non-methylation,and quantitative analysis of m6A in RNA or DNA by Bst DNA polymerase.A site-specific determination of m6A in cancer cells and HCC tissues ha been realized using this method.It was the first time to reveale the different levels of methylation in human rRNA between hepatic cancerous and paracancerous tissues.