Studies On Analytical Methods For The Detection Of Messenger RNA And MicroRNA Based On Nucleic Acid Amplification

The basic process of life-gene expression,that is,genetic information encoded in DNA is transcribed to messenger RNA(mRNA)by RNA polymerases,and mRNA is translated to protein by ribosomes,is the central dogma of molecular biology.With the completion of Human Genome Project(HGP)and rapid developing of next generation sequencing(NGS)technologies,the basic sequence information in DNA and RNA has been read out.However,further studies have indicated that basic process of life is more complicated than the central dogma,such as,individual organism which has the same genome is different each other.The same genome could transcribe to different mRNAs,and congener protein has a variety of functions in different tissue or cell.The above evidences show that mRNA expression not only exhibits individual difference but also spatiotemporal difference,and these differences originate in complex and precision gene expression regulation of organism.Therefore,the study of gene expression regulation is a key which can reveal more biological mysteries.MicroRNAs(miRNAs)were discovered in recent years,a class of endogenous,non-coding,and single-stranded small RNA molecules.MiRNA can inhibit protein translation and induce mRNA degradation,and plays an important role in gene expression regulation.This is not only supplement of central dogma,and opens a new chapter in the study of gene expression regulation.In this paper,by mean of nucleic acid amplification technologies,we established simple,rapid,high specificity,and high sensitivity methods for detecting mRNA and miRNA,which have an important significance for in-depth understanding of gene expression regulation,the study of genetic diseases,early diagnosis and treatment of the cancer,and the mechanism research of carcinogenesis.(1)In the second chapter,we have developed a sensitive,simple,and enzyme-free assay for detection of microRNAs(miRNAs)by means of a DNA molecular motor consisting of two stem-loop DNAs with identical stems and complementary loop domains.In the presence of miRNA target,it can hybridize with one of the stem-loop DNA to open the stem and to produce a miRNA/DNA hybrid and a single strand(ss)DNA,the ssDNA will in turn hybridize with another stem-loop DNA and finally form a double strand(ds)DNA to release the miRNA.One of the stem-loop DNA is double-labeled by a fluorophore/quencher pair with efficiently quenched fluorescence.The formation of dsDNA can produced specific fluorescence signal for miRNA detection.The released miRNA will continuously initiate the next hybridization of the two stem-loop DNAs to form a cycle-running DNA molecular motor,which results in great fluorescence amplification.With the efficient signal amplification,as low as 1 pmol/L miRNA target can be detected and a wide dynamic range from 1 pmol/L to 2 nmol/L is also obtained.Moreover,by designing different stem-loop DNAs specific to different miRNA targets and labeling them with different fluorophores,multiplexed miRNAs can be simultaneously detected in one-tube reaction with the synchronous fluorescence spectrum(SFS)technique.(2)The ability to dissect cell-to-cell variations of microRNA(miRNA)expression with single-cell resolution has become a powerful tool to investigate the regulatory function of miRNAs in biological processes and the pathogenesis of miRNA-related diseases.In the third chapter,we have developed a novel scheme for digital detection of miRNA in single cell by using the ligation-depended DNA polymerase colony(Polony).Firstly,two simple designed target-specific DNA probes were ligated by using individual miRNA as the template.Ligated DNA probe acted as Polony template that was amplified by PCR process in the thin polyacrylamide hydrogel.Due to the covalent attachment of PCR primer on polyacrylamide matrix and the retarding effect of the polyacrylamide hydrogel matrix itself,as the Polony reaction proceeds,the PCR products diffused radially near individual template molecule to form a bacteria colony-like spots of DNA molecules.The spots can be counted after stained the polyacrylamide gel with SYBR Green I and imaged using a microarray scanner.Our Polony-based method is sensitive enough to detect 60 miRNA molecules,meanwhile,the new strategy has the capability of distinguishing singe-base difference.Due to its high sensitivity and specificity,the proposed method has been successfully applied to analysis of the expression profiling of miRNA in single cell.(3)In the forth and fifth chapter,we report a simple and universal strategy,ligation-based loop-mediated isothermal amplification(LAMP),for highly sensitive miRNA and mRNA anaylsis at single-base resolution.Two stem-loop DNA probes are strategically designed,which can be specifically ligated by directly using mRNA or microRNA as the templates with catalysis of RNA ligase.The ligated double stem-loop DNAs subsequently initiate the rapid auto-cycling strand displacement DNA synthesis at a constant temperature,resulting in exponential and faithful amplification with low background and non-specific amplification.So that the proposed assay can be used as a general strategy for detection and quantification of mRNA and miRNA.