The Loop-mediated Isothermal Amplification Technique Based On Stem-loop Structure Primers Detects MicroRNA In Single Cells

MicroRNA(miRNA),a class of small non-coding and endogenous RNA molecules with a length of 19-24 nucleotides,can regulate the process of mRNA translation into protein by either of two posttranscriptional mechanisms:mRNA cleavage or translational repression.It plays important regulatory roles in cell growth,differentiation and apoptosis.A large number of studies have shown that abnormal miRNA expression has relations with the development and progression of human diseases,especially for tumors,and miRNA can be used as new biomarkers for many critical diseases,including cancer.In recent years,miRNA analysis has received significant attention owing to its great importance for fundamental biological research,clinical diagnosis and disease treatment.However,the detection of miRNA introduces many demands due to unique characteristics of miRNA including small size,low abundance,and sequence similarity among family members.In addition,many diseases including cancer,may usually begin with cellular abnormalities in a small minority of cells,especially at their early stages.In consideration of the complex stochastic nature and cell-to-cell fluctuations of gene expression in cells,particularly cancer cells,detection of the average miRNA expression among cells may result in the loss of important information associated with miRNA expression and cell function.With the rapid development of molecular biology and precision medicine,accurately quantitative analysis of miRNA at single cell has become increasingly important.Such kind of analytical methods is significant to revealing the biological functions of miRNA,clinical diagnosis as well as disease treatment.At present,the established methods for miRNA analysis are either with low sensitivity unable to realize single cell analysis of miRNA,or require complex operation processes and expensive experimental instruments.Therefore,to realize single-cell analysis of miRNA by simple operations in homogeneous solution with common instruments in general laboratories is very desirable.In this work,we develop an ultrasensitive method for miRNA detection at single-cell level based on loop-mediated isothermal amplification(LAMP)with two rationally designed stem-loop primers.The sensitivity of nucleic acid amplification-based assays is mainly limited by their amplification efficiency and background signals produced by non-specific amplification.Among the analysis methods of nucleic acid amplification,at present,loop-mediated isothermal amplification is one of the most efficient and sensitive method,which can amplify several copies of target molecules to a detectable level in a short time under constant temperature.However,miRNA cannot be used as a template for LAMP due to its short length.Therefore,we innovatively design a pair of stem-loop DNA primers.First,a stem-loop DNA primer can reverse transcribe along the miRNA by hybridizing with the target miRNA to form the complementary DNA(cDNA).The second stem-loop DNA primer specifically designed can extend and form double-stranded DNA by binding to the cDNA.Through thermal cycle,each target miRNA can produce multiple double stem-loop DNAs,which can initiate LAMP with low background signal and efficient amplification.Thus,a highly sensitive and selective method for miRNA analysis is established.The detection of miRNA in homogeneous solution can reach single cell level with high specificity to discriminate a single-base difference and realize a simple and rapid single-cell analysis of miRNA.The proposed method may serve as a powerful technique to reveal the biological functions of miRNA at single cell level,which is of great importance for miRNA-related biological research and disease diagnosis.