Methods For Detection Of MiRNA Using Nucleic Acid Amplification And Its Application In Biological Sample Detection

Micro RNAs(miRNAs)are one of the most essential markers used in modern molecular biology for the identification of many fundamental cellular processes.Mature miRNAs are single-stranded,small non-coding RNA molecules of 20-25 nucleotides length.miRNAs sequences have recently been shown to be involved in a variety of cellular functions including biological and pathological processes,cancer cell expansion,growth,and differentiation,and apoptosis.miRNA not only regulates a variety of key genes related to cell growth and cell differentiation but also regulate various human diseases including cancer.Many abnormally expressed miRNAs are found in different cancer tissues and participate in the pathogenesis of cancer.For example,miRNAs are found to be up-regulated or down-regulated in breast cancer,lung cancer and colon cancer,and other human B lymphomas.It can be used as a tumor suppressor gene(miR-200 a,miR-15 a,let-7,etc.)to downregulate the activity of proto-oncogenes;it can also be used as an oncogene(miR-155,miR-175 p,miR-21,etc.)down-regulate the activity of tumor suppressor genes.Therefore,in the diagnosis and prognosis of cancer,miRNA is expected to become a promising biomarker for clinical diagnosis and treatment.High specificity and high sensitivity detection of miRNA is of great significance.However,miRNAs have short sequence lengths,large single-cell copy number variations,low in vivo abundance,and high sequence similarity within the same family.These endogenous characteristics bring great challenges to in vivo miRNA detection which limit miRNA detection.The traditional methods for detecting miRNA are Northern blotting,microarray,and stem-loop primer reverse transcription real-time fluorescence quantitative PCR technology(Stem-loop RT-q PCR).However,each method has its own limitations and disadvantages,it is particularly important to develop new,highly sensitive,and specific miRNA detection methods.Part ⅠQuantitative detection of miRNA plays an important role in the research of cancer pathogenesis and treatment methods.We proposed a miRNA detection method based on the self-assembly amplification of phosphorothioated(PS)modified chains.In this method,two stem-loop structure DNAs(linker A and Linker B),each containing a part of the target miRNA recognition sequence,were designed.Part of stem-loop structure(Linker A/B)were PS modified,which will cause the structural instability of the stemloop structure during nucleic acid amplification to form a self-assemble folding.In the presence of the target miRNA,Linker A/B will form a ternary complex,which acted as a dumbbell-shaped DNA strand for self-assembly amplification with T4 DNA Ligase and Bst 2.0 DNA polymerase.PS modification lowered the thermal stability of doublestranded DNA,and promoted the self-folding of the hairpin sequence at the 3’ end to trigger the amplification reaction.This reaction can produce long fragments of DNA without the participation of external primers.The results shown that this method has the advantages of simple operation and high selectivity.The linear range of this method is 300 f M-30 n M under the optimal reaction conditions.The sequence of single nucleotide difference also can be recognized.Part ⅡIn the first part,we developed a miRNA detection method based on SAA.Although this method has many advantages,its sensitivity and long detection time still needs to be further improved.In this part,we designed two stem-loop structure DNA Linker A/B which was from LAMP and can hybridize with miRNA when the target miRNA appeared.A stem-loop structure which was used as dumbbell-shaped amplicon was formed with Splint R Ligase.With the adding of BIP/FIP and Bst 2.0 DNA polymerase,LAMP reaction was carried out.Compared with SAA,LAMP greatly improved the amplification efficiency.Finally,we added the double-stranded DNA fluorescent dye Evagreen to detect the amplification product to achieve the quantification of the target miRNA.This method can detect miRNA in a linear range of seven orders of magnitude,with a detection limit of 100 f M.Part ⅢIn the second part,we established a highly sensitive and specific LAMP amplification-based miRNA detection method.Considering that the signal of Evagreen and other nucleic acid dyes are from all double-stranded DNA,non-specific amplification products will also lead to increased fluorescence intensity.Thus,the false positive results will affect the accuracy of the quantification.Therefore,we designed a sequence-specific fluorescent signal probe to further improve the detection method.A large number of amplicons will be generated during the LAMP amplification reaction.These amplicon sequences with many stem-loop structures can be divided into four types.We chose a stem-loop structure as the recognition unitto to design MB signal probe.When the stemloop structure was produced in the amplification product,the MB probe will be turned on to emit a fluorescent signal to achieve quantitative detection of target miRNA.We also tried OSD signal probe based on strand displacement,and selected MB as the signal probe of the detection method according to the experimental results.This new signal probe has high specificity and selectivity,while greatly reducing the background signal.In order to further improve the sensitivity of the method,we also optimized the design of the LAMP system.In the previous study of the LAMP detection method,we found that PS modified region of LAMP primer would reduce phase transition temperature of the intermediate LAMP product loop,thereby forming self-assembly amplification and enhancing the efficiency of LAMP amplification.We established a simple and fast miRNA detection method,the detection limit of this method reaches 1 a M,can be used to detect blood samples of tumor patients to study the abnormal expression of miRNA as a new biomarker for tumorigenesis,and analyze with other markers such as gene mutations to improve the specificity of tumor diagnosis.It can also be applied to the in vivo metabolism of new miRNA drugs.