Establishment and application of a standardized fluorescent quantitative PCR method for detection of cellular IDO gene expression
In order to establish a rapid,accurate,sensitive and intuitive method for detecting IDO gene expression,a real-time fluorescence quantitative PCR detection method was developed by designing specific primers,preparing IDO plasmid stan-dards,and constructing a standard curve.The results showed that the IDO gene fragment was successfully cloned into the vector,forming the recombinant plasmid pSin-flag-IDO,and the standard curve exhibited a good linear relationship.Amplifi-cation identification was performed using both the constructed fluorescence quantitative PCR detection method and the con-ventional PCR detection method(as the current detection standard).The lowest detection limit of conventional PCR was 660 copies/µL,while that of fluorescence quantitative PCR was below 66 copies/µL,indicating a higher sensitivity of the fluores-cence quantitative PCR detection method.The inter-group repeatability test was conducted on five concentrations of the standard,with all coefficients of variation being less than 10％,demonstrating good repeatability of the detection method.The establishment of this method can make the detection of induced IDO expression more accurate,which is beneficial for the improvement of the group standards for human mesenchymal stem cells.
Gene expression of IDO(Indoleamine 2,3-dioxygenase)Fluorescent quantitative PCRDetection method
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