| MicroRNA is a small endogenous non-coding single-stranded RNA with a length of about22 nucleotides.Mi RNA interacts with the 3’-untranslated region(UTR),5’-untranslated region(UTR)or coding sequence(CDS)region of the target gene to mediate gene silencing at the post-transcriptional / translational level and reduce the stability of m RNA,leading to the degradation of m RNA and the inhibition of protein translation.Mi RNA can control the activity of more than 50% of human coding genes and plays a key role in a variety of biological processes.In recent years,more and more evidences demonstrate that the miRNA are closely related to human diseases,and the abnormal expression of miRNA is observed in almost all types of tumors,including lung cancer,cervical cancer,and breast cancer.Thus,miRNAs can be used as the biomarkers for tumors.Notably,multiple miRNAs can jointly regulate a single gene function,and a single type of disease may be associated with the deregulation of various miRNAs.Detection of only one kind of miRNA is likely to cause false negative / positive results.Consequently,development of sensitive methods with the capability of simultaneously detecting multiple miRNAs is highly desirable in the field of biomedical research,early clinical diagnosis and anticancer drug discovery.Lung cancer is one of the most common cancers worldwide,and its survival rate is hugely influenced by stage at diagnosis,with the early 5-year survival rate being 92% and the 5-year survival rate of terminal cancer being only 0-20%.Because its early symptoms are not obvious,most of patients have already been in the late stage when they are diagnosed.Therefore,it is of great important to find new biomarkers for early diagnosis and cancer therapy.Studies have shown that miR-155 is overexpressed in lung cancer and its high expression is associated with poor prognosis in lung cancer patients.In addition,miR-21 as an oncogene is upregulated in various cancers including lung cancer.In this research,we choose miR-155 and miR-21 as the model miRNAs.We develop a triple signal amplification system for simultaneous detection of multiple miRNAs at single-molecule level based on the combination of strand displacement amplification(SDA),apurinic / apyrimidic endonuclease 1(APE1)-assisted cyclic cleavage,rolling circle amplification(RCA)and single-molecule detection.In the presence of two target miRNAs,they can hybridize with their complementary linear templates to initiate SDA reaction,generating a large number of triggers,respectively.Then the triggers can be paired with their AP probes to initiate APE1-assisted cyclic cleavage-mediated signal amplification,generating specific primers,respectively.The resultant two primers can hybridize with their respective circular templates to initiate RCA reactions.Because the two circular templates are specially designed with each containing only three bases,Cy5-labeled d CTP(Cy5-d CTP)and Cy3-labeled d GTP(Cy3-d GTP)can be specifically embedded in the RCA amplification products.After the removal of excess Cy5-d CTP and Cy3-d GTP by magnetic separation,both Cy3 and Cy5 fluorescent molecules can be accurately quantified by total internal reflection fluorescence(TIRF)-based single molecule detection,with Cy5 indicating miR-155 and Cy3 indicating miR-21.The single molecule technology exhibts high sensitivity with a detection limit of miR-155 is 25.73 a M,and the detection limit of miR-21 is 45.71 a M.Moreover,it possesses good selectivity with no interference between two targets,and it can even distinguish the mismatch of single base.Furthermore,this method can be used for the simultaneous detection of multiple miRNAs in real samples,and it can discriminate patients tissue samples from normal tissue samples.Importantly,this method is ingeniously designed for simultaneous detection of multiple miRNAs without the involvement of any specially labeled detection probes.It provides a new platform for ultrasensitive detection of multiple miRNAs,with great promise in early lung cancer diagnosis. |
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