Fro Sufferer's Quadruple Heart Mirna Expression Analysis And The Establishment Of General Micrornas Taqman Probe Detection Method And Application

Part OneMicroRNAs (miRNAs) are endogenous 22-25 nucleotides that are processed from larger hairpin precursors, whose function is as regulator of gene expression. Pri-miRNAs which often contain 7mGpppG and polyA are transcript by RNA polymerasesⅡin nucleolus. Then the stem loop intermediate pre-miRNAs have been liberated performed by Drosha RNaselll endonucleas. These pre-miRNAs can be transported to cytoplasm by RAN-GTP and exportin-5, where mature miRNA is cleavage by Dicer from one arm of them. Mature miRNA combines with the complementary base of mRNA to regulate gene expression through inhibiting translation or degrading specific mRNA.MiRNAs are believed to participate in variedly regulation approaches, including cell development, ulation of hematopoietic lineage differentiation, cardiovascular biology and human tumorgenesis.Tetralogy of Fallot (TOF) is the most common heart defect in children which occurs at approximately 7%-8% of congenital heart defects (CHD). Infants with this abnormality develop signs of the condition very early in life, including Pulmonary stenosis, Overriding aorta, ventricular septal defect (VSD) and Right ventricular hypertrophy. Although their etiology is often poorly understood, most are considered to arise from multifactorial influences, including environmental and genetic components. MiRNAs have been paid great attention in resent research. But to our best of knowledge, microRNA expression on TOF in human has not been previously reported.ObjectionFind out the TOF-associated miRNA and determine the target gene.Methods and results(1) MiRNAs microarray was used to detect miRNA expression of 3 normal and 5 TOF heart tissues. Among the screened microRNAs,40 signals have different experssion between normal persons and TOF children.(2) Expression of all the selected microRNAs by q-RT-PCR from more TOF patients (n=26) and normal persons (n=6). The analysis confirmed that 13 microRNAs have differently expression patterns and contain 10 up-regulated and 3 down-regulated. (3) Since the control samples’ages were older than the TOF samples, there may have differences in miRNAs expression with age changes. To avoid this false positive result,2-3month and 10 days C57 mouse heart tissues was used to correct the outcome acquired by q-RT-PCR. There are 8 miRNAs which growth and development have effect on.(4) To analysis the biological significance of miRNA deregulation, we use bioinformatics tools for target gene prediction of miRNAs verified by q-RT-PCR from the Candidate gene library of congenital heart disease and verified in vitro by luciferase array.Conclusions:The miR-363, miR-424, miR-424*, miR-181c and miR-181d were up-regulated in Tetralogy of Fallot and luciferase activity of NF1 and HAS2 3’-UTR was inhibited 50% and 40% by mir-424, while PAX3 and HAS2 3-UTR was inhibited 40% by mir-363.Part TwoThe gene regulation mechanisms of miRNAs have had great advancement since the discovery of these small non-encoding molecules. Therefore, methods for identification and quantified detection of miRNAs are important in research.The conditional miRNA detection contains two:1) Northern blot:this method often needed plentiful RNA and isotope.2) Quantitative real-time PCR (q-RT PCR) assays:this method has been proven to be a sensitive and specific tool for miRNA expression, which contains probe-based (TaqMan probe array) and probe-less (SYBR Green array) methods. SYBR Green is a dye that binds to all double-stranded DNA molecules, and it may overestimate the quantity of target because the formation of primer dimer can give the false results. In contrast, the TaqMan probe may target the DNA with a particular region based on its probe hydrolysis. Even it had high specificity for miRNA detection, but it is expressive to design each target probe in abundant experimental use. Since either of them has shortcomings, new method to detect miRNAs are needed for the research.Objection:Send up a universal TaqMan probe method to detect miRNAs.Methods and results (1) The TaqMan probe was designed and according to the modified anchor primer and in front of the localization of the universal reverse primer.(2) To examine the feasibility of the novel, specific TaqMan probe, miR-126 was used as a positive control to detect.(3) MiR-1-1 and miR-133a was used to determine the miRNA expression level in tissue by the modified TaqMan probe.(4) Compared with SYBR Green method, it is not affect by the primer dimer as the traditional TaqMan probe array and would precise quantification depend on the template concentration with a larger region.(5) The modified TaqMan probe was used to discover miRNA expression profile in five different tissues.ConclusionsWe present a modified TaqMan probe q-RT PCR method for easy and accurate monitoring of miRNAs. This method bases on the modified polyA RT-PCR and use universal TaqMan probe to detect miRNAs.