| Background and objectiveMicroRNAs?miRNA?are a group of single-stranded non-coding RNA molecules with a length of 19825 nucleotides.As regulators,microRNAs regulate gene expression by binding to the 3'-untranslated regions of messenger RNA?mRNA?through a dual-mechanism:translational repression and target degradation.Since their discovery in Caenorhabditis elegans in 1993,more than 17,000 microRNAs have been identified experimentally or by bio informatics across different species including human.They play an important role in many biological and pathological processes and have been confirmed to have a close relationship with the development of many different diseases,such as viral diseases,cardiovascular diseases,immune disorders,and cancers.On the other hand,a large number of recent reports have shown that microRNAs were not only found in the cells but also in various body fluids such as plasma,saliva,urine,tears,semen,and amniotic fluid.Therefore,microRNAs are considered as promising candidates for next generation biomarkers specific for the diagnosis and prognosis of corresponding diseases.Although miRNAs can be find in many kinds of biological samples,not every sample can be used as the ideal analysis object.An ideal biomarker should meet the following two characteristics.First and foremost,it should be easy to obtain and does not require expensive equipment or complex process.Secondly,it also should be reproducible.Because it is critical for real-time and continuous monitoring of disease.Thus,currently,many researchers shift their focus from cultured cells and soled tissues to blood,and have found large numbers of diseases associated C-miRNAs?circulating microRNAs?.While,unlike the widely used protein biomarkers,C-miRNAs are not in free form in the blood.They usually exist in blood in the following three forms:?1?incorporation into microparticles?microvesicles,exosomes,and apoptotic bodies?;?2?formation of a ribonucleo-protein complex with Argonaute proteins;?3?lipoprotein complexes.All of this means that a high-efficiency protocol for microRNA isolation from serum sample is particularly important.Currently,several methods have been developed for the extraction of miRNA from biological sample,such as the TRIzol based isolation technique,the acid phenol based extraction technique and some commercial isolation kits.Among them,the TRIzol based isolation technique has become one of the most widely used methods for its classic,reliable,and economic.Because,it is generally used to isolate miRNA from tissue samples or cultured cells.When it is used for serum samples,the isolation efficiency cannot meet the requirement of microRNA research in most cases.In recent years,many kinds of microRNA isolation kits have been developed and aroused great interest because of their time-saving advantage and better extraction efficiency.However,the high cost restricts their widespread and multiple use.Thus,in this study,after in-depth investigation of the principle of the TRIzol-based technique and further practice of RNA extraction,we optimized the original TRIzolbased technique to adapt it to the extraction of microRNA from serum sample.Then,the improved TRIzolbased method and the traditional one were both used to extract miRNA from serum samples to observe the effect of improvement.In addition,we also evaluated our improved protocol by comparing its isolation efficiency with the other two available isolation protocols.We hope that that our method is valuable for researchers interested in developing serum microRNA biomarkers for diagnosis or prognosis,especially for those researchers to whom a commercial kit is not available.On the other hand,it also very important to establish a good method for the quantitative analysis of microRNA.Because,In comparison with other nucleic acids including DNA and messenger RNA,the unique characteristics of miRNAs,such as small size,sequence homology among family members?even single-base mismatch?,and low abundance in biological samples,make accurate analysis a great challenge.In the past decade,a variety of effective methods have been developed for miRNA detection,among which northern blotting is regarded as the gold standard,although has limitations such as poor sensitivity,large sample consumption and a tedious experimental protocol.The microarray-based technique is another conventional assay which has the advantage of high throughput and multiplexing capacity,but poor sensitivity and a lengthy hybridization time restrict its wide application.As for the most widely method?RT-PCR method?,it has the advantage of high sensitivity and specificity.But,unfortunately,it requires precise temperature control and the essential step of reverse transcription,which increases the experimental time and risk for RNase contamination.Therefore,the development of a rapid,convenient and sensitive miRNA detection method is still in great demand.Thus,in this study,based on DR-CPs and DSN-assisted dual signal amplification,we have developed a rapid,highly sensitive and specific miRNA detection strategy.After that,under the optimized condition,the sensitivity and specificity and specificity of this new method were estimated.We hope to provide a good method for miRNA detection,which hods great potential for further application in biomedical research and in the clinical laboratory.Methods1.Optimization of the original TRIzol-based technique improves the extraction of circulating microRNA from serum samples1.1 After in-depth study of traditional TRIzol method extracting steps and the physical and chemical characteristics of miRNA,we optimized the original TRIzolbased technique to adapt it to the extraction of miRNA from serum sample.1.2 Four RNA isolation methods,including the original TRIzol-based method,two partly improved methods and the improved TRIzolbased method were used to extract miRNA from four preprocessed serum samples.Then,the extraction efficiency were evaluated respectively,thereby verifying the effect of protocol optimization.1.3 To determine the isolation efficiency of the improved TRIzol based method,three isolation methods including the improved method,miVanaTM miRNA Isolation kit and the single-step acid phenol extraction method were compared for isolation of microRNA from human serum sample.2.Ultrasensitive and selective detection of microRNA based on DNA/2-OMe-RNA chimeric probes assisted duplex-specific nuclease signal amplification2.1 Primer Premier 5.0 software was used to design all the oligonucleotide probes used in this study.Then,all the oligonucleotide sequences were synthesized by Takara Biotechnology Co.Ltd.2.2 Products of the DSN enzymatic amplification reaction were analyzed by 3%agarose gel electrophoresis,thereby verifying the effects of DSN on DNA/RNA and DNA/2-OMe-RNA hybrid duplexes.2.3 Streptavidin-coated MMs were utilized as vehicles to load DR-CPs through the interaction between biotin/streptavidin.Then,confocal microscope was used to characterize the functionalized microspheres and verify the successful conjugation of biotinylated oligonucleotides and streptavidin-coated MMs.2.4 To obtain the best performance,the reaction temperature,the concentration of Taqman probes,and the amount of DSN were optimized in turn.2.5 Under optimal conditions,the sensitivity of the proposed method was assessed by measuring miRNA-21 at various concentrations.Then,the detection limit and the linear range of this method was calculated.2.6 To investigate the detection specificity of this strategy,several RNA sequences,including miR-21?target miRNA?,M1?one-base-mismatched,middle?,M2?one-base-mismatched,close to one end?,M3?two-base-mismatched?,M4?there-base-mismatched?and miR-141 as a random sequence,were selected and detected under the same conditions.2.7 Total RNA extracted from three kinds of cancer cell lines were analyzed using the developed strategy and qRT-PCR,thereby verifying the performance of the developed method for the detection of miRNA in real sample.Result1.Optimization of the original TRIzol-based technique improves the extraction of circulating microRNA from serum samples1.1 After establishing an optimized TRIzol-based method,we compared the performance of the improved TRIzol-based technique with the improved method A,improved method B,and the original TRIzol-based method.The result suggested that the optimized TRIzol-based method can obviously improve the purity and output of extracted RNA and then can decreased cycle number of the following PCR analysis.1.2 The improved TRIzol-based technique was also compared with two usually used method.The result show that the improved method out-performs the other two isolation methods we tested,at least for isolation of microRNAs from serum sample.2.Ultrasensitwie and selective detection of microRNA based on DNA/2-OMe-RNA chimeric probes assisted duplex-specific nuclease signal amplification2.1 The agarose gel electrophoresis was performed to analyze the effects of DSN on DNA/RNA and DNA/2-OMe-RNA hybrid duplexes,respectively.The result show that DSN has no significant difference in its preference for DNA/RNA or DNA/2-OMe-RNA hybrid duplexes.2.2 Characterization of functional microspheres was carried out by the confocal microscope was used.The result suggest that the biotinylated oligonucleotides can successful and efficient fix to the surface of well-dispersed microspheres.2.3 The Optimization experiment show that the best suitable reaction temperature is 60?,the best addition quantity of DSN is 0.2U and the optimal concentration of Taqman probe is 300nM.2.4 Under optimal conditions,the fluorescence intensity exhibits a positive linear correlation with the concentration of miRNA-21 within the range of 10 fM to 10 pM.Meanwhile,the proposed strategy exhibits high specificity in discriminating cognate miRNAs,and enables the detection of miRNA concentrations as low as 7.3 fM.We further applied this assay to miRNA quantification in cancer cells to confirm its applicability.The results obtained with the proposed method were consistent with those of qRT-PCR and were also in good agreement with previous reportsConclusionIn conclusion,we have established an economic and efficient protocol for microRNA isolation from human serum and the developed protocol is able to extract high quality microRNAs with enough yield for real-time PCR-based profiling experiments.Compared to the other two widely used methods,we found that our method had better performance in terms of decreased cycle number of the following PCR analysis and deserved trust.We believe that our method is valuable for researchers interested in developing serum microRNA biomarkers for diagnosis or prognosis,especially for those researchers to whom a commercial kit is not available.On the other hand,based on DR-CPs and DSN-assisted dual signal amplification,we have developed a rapid,highly sensitive and specific miRNA detection strategy.Due to the ultra-high selectivity of DSN and the signal amplification efficiency of DR-CP-assisted dual signal amplification strategy,the proposed new method can detect a concentration of miR-21 as low as 7.3 fM and exhibits an excellent discrimination ability even for the differentiation of highly similar miRNA sequences with a single base difference.These features,together with other advantages such as operational convenience,a fast protocol and simple design,make this method a promising candidate for miRNA detection and early disease diagnosis.Therefore,we believe that our novel sensing strategy will offer a new platform for highly sensitive and specific detection of miRNA and holds great potential for further application in biomedical research and early clinical diagnosis. |
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