Development and optimization of an exosomal miRNA extraction method
Objective To develop and optimize the extraction method of exosomal miRNA and compare it with traditional methods.Methods The exosomal miRNA of MSC,NK and CIK cells was used as the research subject.The removal efficiency of genomic DNA from exosomal miRNA by gDNA removal column was detected by UV spectrophotometer and agarose gel electrophoresis.The lysates(exosome G2 lysate and exosome miRNA lysate)and aggregation reagents(absolute ethanol and isopropanol)were optimized by using concentration,purity and gene expression level(Ct value)as evaluation indexes.Exosomal miRNA of MSC,NK,CIK cells and healthy human serum was extracted by the developed method,Trizol method and Trizol magnetic beads method,and detected by RT-qPCR.Results The gDNA removal column effectively removed genomic DNA from exosomal miRNA.The concentrations of exosomal miRNA extracted from MSC,NK and CIK cells by using exosome miRNA lysate were significantly higher than those by using exosome G2 lysate(t=6.358,P=0.020).The purity of miRNA samples extracted by exosome G2 lysate was low,and there was foreign protein pollution,but exosome miRNA lysate effectively removed the pollution.The Ct values of miR-Let-7i,miR-16-1 and miR-150 genes in exosomal miRNA of CIK cells extracted by exosome miRNA lysate were significantly lower than those by exosome G2 lysate(t=30.120,P=0.008).The concentration of exosomal miRNA extracted from MSC,NK and CIK cells by isopropanol was significantly higher than that by absolute ethanol(t=8.567,P=0.010),and the purity was significantly lower than that by absolute ethanol(t=6.214,P=0.020).The Ct values of miR-Let-7i,miR-16-1 and miR-150 genes in exosomal miRNA extracted from CIK cells by two aggregation reagents had no significant difference(t=2.297,P=0.120).Compared with Trizol method and Trizol magnetic beads method,the expression of miR-Let-7i,miR-16-1,miR-Let-7a and miR-150 genes in exosomal miRNA extracted from CIK,NK,MSC cells and healthy human serum by the developed method was more abundant,and the overall Ct value was lower.The dissolution peak was prominent and sharp,exhibiting a single main peak.Conclusion The exosomal miRNA extracted by the developed method has high concentration and purity with stable Ct value,and the method has high sensitivity and good specificity.This study lays a foundation for further research on exosomal miRNA.
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