Experimental Research On Molecular Beacons Used For Detection Of Endogenous MRNA In Living Cells

Background: As a new type of nucleic acid fluorescent probes,Molecular beacons have real-time supervision,high sensitivity and high specificity when it is applied in vitro.However,it has little applied in living cells in the body,especially in the field of zebrafish research has yet to see the application of molecular beacons experiment reports.Try applying molecular beacons in the zebrafish embryos for research has a good value for further application of molecular beacons in animals such as the gene detection and tracking.Through the application of the molecular beacons in living cells in the body,it can provide potential method for its further application in zebrafish embryos in vivo and other level of genetic testing.Materials and Methods: 1 the experimental study of Molecular beacons in vitro 1.1 Stability Experiments The stability of the molecular beacons sequence contains two aspects,that is the integrity and the stability of the fluorescence intensity.Temperature is an important condition which influence the stability of the molecular beacons.We put the molecular beacons in water bath for different time,through the agarose gel electrophoresis(AGE)to evaluate the temperature affection to the stability of the molecular beacons,and test the temperature effect on the stability of the fluorescence intensity by melting curves.1.2 Specificity and sensitivity experiments The specificity and sensitivity are two key factors to determine the application values of the molecular beacons.We then checked the specificity and sensitivity of this MB through binding with synthetized target sequence assay.Firstly,we use the melting curves to identify the specificity of the molecular beacons by combined with the target sequences.Secondly,we identify the relevance between the signal of molecular beacons intensity and concentration of target genes.2 The experimental study of molecular beacon in living cells After verify the stability and specificity of molecular beacons in vitro,we continue to verify the molecular beacon's specificity of the combination of m RNA in living cells.We first transfected eGFP to the Living 293T cells,and then transfected the molecular beacons target eGFP into the 293T cells expressed eGFP and normal 293T cells respectively.Acquire fluorescent images of the cells.3 The experimental study of molecular beacon in vivo The specificity and sensitivity of the molecular beacons in combination with target genes in vivo is our further research direction.We respectively microinjected the molecular beacons and its exogenous targets single stranded into the embryos of wild type zebrafish,and then observed the fluorescence expression.We microinjected the molecular beacons and its targets single stranded during 1-cell period,observe the fluorescence expression.RESULTS 1 The stability of the molecular beacons in vitro The AGE results showed that the eGFP MB was stable under 37 ? within 48 hours,and the fluorescence intensity difference has no statistical significance(p > 0.05).It shows that MB can maintain its own sequence and stability within 48 hours in vitro.2 The specificity and sensitivity of the molecular beacons in vitro The results showed that only in the presence of target sequence,molecular beacon's signal significantly enhanced,and the signal intensity was positively correlated with the concentration of target genes.3 The molecular can specifically bind to the endogenous m RNA in living cells The eGFP MB could specifically bind to target gene.The fluorescence of MB could not be detected with low concentration of target gene.Living 293T cells have fluorescence expression after transfection of eGFP and eGFP MB.It showed that the molecular beacon can used to detect endogenous m RNA in living cells.4 The molecular can bind to the target sequences in zebrafish embyos The fluorescence of MB also could be observed in 1-cell stage zebrafish embryos injected with eGFP MB and its taget sequesce.It shows that the MB in zebrafish embryos are stable and can be specifically bind to target gene,that it means the MB can be applied in zebrafish embryos and other levels of endogenous gene detection and tracking in vivo.Conclusion: 1.We design and synthesis of the molecular beacons of eGFP.They are stable,and have good specificity and sensitivity combined with its target genes in vitro.2.The molecular beacons can specifically bind to target genes in living cells,it means the molecular beacons can be used in the detection of genes within living cells.3.The MB in zebrafish embryos are stable and can be specifically bind to target gene,it means the MB can be applied in zebrafish embryos and other levels of endogenous gene detection and tracking in vivo.4.Furthermore,by tracking the specific m RNA of molecular for cardiac in vivo,we can track the spectrum of heart at very early stage,the purpose of the early development of the heart study.