Study On The Amplification Efficiency Of Plasma Exosome Mirna In Cancer Patients Based On Cdna Library

miRNA is a non-coding single-stranded RNA molecule with a length of about 22 nucleotides.It has been proved that miRNA is involved in the formation of various tumors.Controlling and inhibiting the formation of miRNA can reduce the formation of tumors,but miRNA is in the cell The amount of expression is very small,and the mechanism of action of miRNA in tumors is not completely clear.In this study,two methods were used to construct miRNA cDNA library templates for plasma exosomes of cancer patients,and to explore the factors affecting the amplification efficiency of RT-qPCR reaction system.Templates were constructed by connecting aptamers at the 3'and 5'ends of the miRNA and polyA and 5'at the 3'end.The amplification efficiency was analyzed by RT-qPCR,using enzyme digestion and magnetic beads The separation method removes non-specific amplification in the amplification.The main experimental results are as follows:In the miRNA stability experiment,the miRNA was stored under different temperature,pH and time conditions,the Ct value was obtained by RT-qPCR reaction,the miRNA concentration was quantified by the standard curve,and the optimal storage was optimized by quantitative analysis of different influencing factors Conditions,short-term storage can be at 4?,long-term storage at-80?,storage buffer is neutral.In the template construction experiment,the first method was to connect a piece of aptamer to the 3' end and 5'end of miRNA respectively,and verify the successful construction of the template by denaturing polyacrylamide gel electrophoresis,and conduct RT-qPCR experiment,the experimental results appeared Non-specific amplification curve.The second method is to connect PolyA at the 3'end and connect an aptamer at the 5'end.The modified polyacrylamide gel electrophoresis verifies that the template is successfully constructed,and the qPCR experiment results show a non-specific amplification curve.Both methods have low amplification efficiency due to non-specific amplification.In the non-specific amplification and removal experiment,the non-specific amplification was derived from the unligated aptamer at the 5'end,and the 5'end excess aptamer was removed by phi29 DNA polymerase,nuclear exonuclease T(EXOT enzyme)and magnetic beads.For example,phi29 DNA polymerase and exonuclease T have low degradation efficiency and specificity.9%PEG magnetic bead buffer precipitates large fragments,15%PEG magnetic bead buffer precipitates target fragments,and magnetic beads separation efficiency is higher.In cell and cancer patient plasma exosome validation experiments,successfully constructed RT-qPCR reaction templates for 293T cell total RNA and cancer patient plasma exosome miRNA.