A quantitative detection method for exosome miRNA

Abstract

The invention provides a quantitative detection method of exosome miRNA (micro ribonucleic acid). The quantitative detection method comprises steps as follows: exosome total RNA extraction, connection of 3'-terminal and 5'-terminal specificity unique tagged connectors, reverse transcription synthesis of cDNA (complementary deoxyribonucleic acid), PCR (polymerase chain reaction) amplification, screening, purification and sequencing of a miRNA library on a computer, information analysis of an error correction algorithm and the like. According to the quantitative detection method, all original templates are distinguished on the basis of marks of the specificity unique tagged connectors, preference of molecules is avoided during amplification, miRNA molecules with the copy number lower than 10 can be detected precisely, the specificity is high, and the method can be used for detection of tumor derived exsome miRNA in the fields of neurodegenerative diseases, cardiovascular diseases, reproductive health and the like, can provide a basis for early screening of diseases, can also be applied to the field of precise quantitative detection of other small RNA and has a wide application prospect and high market value.

Description

technical field [0001] The invention belongs to the technical field of high-throughput detection of small molecule RNA, and in particular relates to a quantitative detection method of exosome miRNA. Background technique [0002] Exosome (Exosome) is a kind of membranous vesicle secreted outside the cell after late endosome (also known as multivesicular body) fuses with the cell membrane. Under the electron microscope, its appearance is a characteristic "disk". Shaped small bodies, with a diameter between 40-100nm, are secreted by living cells and can be secreted by almost all types of cells. The nucleic acid, protein, and lipid components carried by them have the specificity of their source cells and participate in the regulation of many Important cell physiological activities, such as immune response, apoptosis, angiogenesis, inflammatory response, coagulation and other processes, so it has important research significance in related diseases, including neurodegenerative dis...

AI Technical Summary

Problems solved by technology

[0004] At present, there are only a few reports on the miRNA in exosomes in the early diagnosis of tumors, and most of the studies are based on the total concentration of miRNA in exosomes, quantitative PCR (Q-PCR) and microRNA chip technology, and more Focus on miRNA expression and quantification, limited to detection of known miRNA expression profiles

In addition, miRNA high-throughput sequencing is also undergoing related attempts. Although its high-throughput and the exploration of new microRNA markers have played a huge role in promoting the development of early diagnosis of tumors, there is a systematic bias in traditional library construction. And a higher error rate, which affects the detection sensitivity, accuracy and related applications

Images