| Thyroid cancer is one of the most common malignant tumors in the neck.In the past few decades,the incidence of thyroid cancer has steadily increased.Papillary thyroid carcinoma(PTC)is the main subtype of thyroid cancer,accounting for 80% of all malignant thyroid tumors.Although most patients with PTC can obtain well therapeutic effects after surgery and postoperative radioiodine therapy,10% of patients still have tumor recurrence or distant metastasis,or even die.With the development of biological technology,a large number of studies have confirmed that MicroRNA(miRNA)as a molecular marker can be used in the clinical diagnosis and treatment of thyroid tumors and has broad prospects.miRNA is a single-stranded non-woven RNA with a length between 19 and 25 nucleotides.miRNA is involved in regulating various biological processes of organisms,such as cell proliferation and differentiation,metabolism,embryonic development,inflammation,tumor formation and programmed cell death.Due to the inherent characteristics of short sequences,low abundance,and high sequence similarity among family members,miRNA is difficult to accurately detect and analyze in actual samples.In recent years,a variety of methods have been developed to detect miRNA,such as Northern blot,microarray method,RT-q PCR,NGS,etc.Although most of these methods can detect miRNAs with high sensitivity and accuracy,they also have some limitations,such as complicated probe/primer/template sequence design,cumbersome detection steps,long analysis time,and expensive reagents.Therefore,we still need to develop a feasible and simple method to quickly quantify miRNAs in actual samples.This article introduces an enzyme-free,sensitive,simple and cheap miRNA detection method based on toeholdmediated strand displacement reaction(TSDR)combined with multifunctional magnetic beads(MB)and flow cytometry fluorescence detection technology.To detect hsa-miR-146b-5p related to PTC.The research content mainly includes:In this study,the difficulty of experimental steps and probe fabrication was greatly reduced by the advantages of large specific surface area of magnetic beads,strong capture ability and rapid separation and collection of signal molecules.Combined with the principle of TSDR and flow cytometry powerful fluorescence analysis capabilities,we have constructed a highly sensitive,specific and convenient and inexpensive miRNA detection method for detecting hsa-miR-146b-5p related to PTC.First,we fixed the double-stranded fluorescent probe with toehold region on the surface of MB.In the presence of hsa-miRNA-146b-5p,hsa-miRNA-146b-5p specifically recognizes the toehold region and the double-stranded fluorescent probe Combines and initiates the TSDR reaction,causing the shorter fluorescent molecular chains to detach from the surface of the MB.Subsequently,a flow cytometer was used to quickly analyze the fluorescence intensity on the surface of the MB,and by detecting the attenuation of the fluorescence signal on the surface of the MB,the concentration of hsa-miRNA-146b-5p in the test solution was calculated.This reaction can be carried out in an enzyme-free environment at room temperature or physiological temperature,which overcomes the potential defects of traditional enzyme-catalyzed signal amplification strategies.Under the optimal experimental conditions,hsa-miRNA-146b-5p has a linear relationship with the fluorescence signal in the range of 5 pmol/L-5000 pmol/L,and the detection limit is 4.21 pmol/L.In addition,this method has excellent specificity and can specifically distinguish the homologous sequences of hsa-miR-146b-5p.This method has been successfully applied to the analysis of hsa-miRNA-146b-5p content in thyroid cells and tissues,and has shown great potential in miRNArelated biological research and disease diagnosis. |
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