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Detection and Imaging of Cancer-related MicroRNA

Author: LiuHaiYun
Tutor: TangBo
School: Shandong Normal University
Course: Cell Biology
Keywords: Cancer MicroRNA Fluorescence Detection Cell Imaging
CLC: R730.4
Type: PhD thesis
Year: 2014
Downloads: 12
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Abstract

MicroRNA (miRNA) is a very active area currently, it is great significance to elucidate theessence phenomenon of life and explain the mechanism of cell behavior and disease, as well asdisease diagnosis, gene therapy drug. Study on microRNA was named the year ’s top ten sciencenews of Science consecutively. Through gene silencing mechanisms, microRNA regulate criticallife processes, such as, cell proliferation, development, differentiation and apoptosis, but studieson the biological function of microRNA still in the exploratory stage. While the expression levelof intracellular microRNA are closely related to major human diseases, especially cancer. Afterfollowing the RNA interference (RNAi), studies on the mechanism of microRNA and humanrelations major diseases will formate an new gene therapy. Since the microRNA is endogenous,it is possible more direct and effective introduce than exogenous. So, the analysis method has acrucial role in biological research of microRNA. MicroRNA analysis methods currently usedmainly Northern Blot, RT-PCR and Microarray. Due to the complexity or sensitivity limits in theoperation process, these methods can not meet the needs of the biological function of microRNAresearch.In order to provide practical and reliable analytical detection technology platform forresearch micoRNA biological functions, to further explore the biological function of microRNA,it is important for microRNA analysis, easy to operate, high-sensitivity analysis, real-timefluorescence imaging of intracellular microRNA. This paper developed some new methods forintracellular microRNA detection and imaging, applications detection technology research, suchas the rolling circle amplification, cyclization amplification, hybridization chain reaction andgraphene.The main contents are as follows:1. A high specific and ultrasensitive isothermal detection of label-free microRNA is achievedbased on P-ERCA reaction. This reaction is initiated by the target microRNA and catalyzed byDNA products generated and accumulated during the reaction. Unlike conventional nucleic-acidamplification reactions such as polymerase chain reaction (PCR), this reaction does not requireexogenous primers, which often cause primer dimerization or non-specific amplification. This experiment is a simple. With the exponential amplification, fluorescence signals can besensitively detected with a remarkable sensitivity of0.24zmol. Moreover, using the reasonabledesigned padlock probe and T4RNA ligase2in the specific ligation, high specificity wasachieved, even single–nucleotide difference can be discriminated. In addition, the proposedstrategy has successfully achieved the detection of let-7a in total RNA sample extracted fromhuman lung cells. The results indicate that the proposed P-ERCA strategy holds a great potentialfor further application in biomedical research and early clinical diagnostics.2. An accurate and sensitive circular exponential amplification assay for microRNAs isdeveloped based on GO-protected target. In this assay, the target microRNA can be protectedfrom enzymatic digestion effectively after non-covalent adsorption on GO surface. Throughrepeated hybridization, extension, enzymatic cleavage and release of triggers, circularexponential amplification for microRNA was achieved. Unlike the microRNA was released andused as new triggers to initiate next cycle in most reported reaction, in this assay, the releasedDNA triggers are more stable and preferred for the sustained reaction. Moreover,multi-molecules binding of the intercalating dye SG to double-stranded DNA induced significantenhancement of fluorescence signal. The circular exponential amplification based ongraphene-protected target microRNA exhibited excellent specificity to discriminatesingle-nucleotide difference and allowed quantification of microRNAs with a sensitivity of10.8fM. The microRNAs analysis in human lung cells was also performed, indicating that thisstrategy will become a reliable and sensitive microRNAs quantification method in biomedicalresearch and early clinical diagnostics.3. Based on graphene and hybridization chain reaction, the method is developed for imagingof living cells, microRNA is sensitive detected by this method in living cells. Graphene as acarrier, hairpin probes H1and H2can be deliveryed into the cell, but also graphene as a paltformfor hybridization chain reaction. It is results that double-stranded DNA and relative highfluorescence intensity concentration producted. This method can be used to observe microRNAimaging of living cell directly. Meanwhile, it is provide a new tool for the early diagnosis andimaging of cancer. 4. A method for imaging of microRNA is developed based on graphene and hybridizationchain reaction simultaneously in living cells. Graphene as a carrier, a hairpin probe can betransported into the cell, but also graphene as platform for hybridization chain reaction.Hybridization chain reaction produced enriched double stranded DNA product, resulting inrelatively high fluorescent. Since the specific of hybridization chain reaction, this method can beused two or more microRNA simultaneous imaging tool in living cell, and for direct observationin different expression levels of microRNA in the cell. It is provide a new tool for the earlydiagnosis of cancer.

Detection and Imaging of Cancer-related MicroRNA

Abstract

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