Real-time and Droplet Digital PCR Analyses for Detection of Vibrio cholerae Gene Expression by Four Gene Quantification Methods
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Abstract:
Gene expression assays are highly important for analyzing gene function and investigating biological mechanisms. Currently, real-time and droplet digital PCR are effective methods for measuring gene expression. sfs, vcc, RecA, hly, and 16S rRNA genes of Vibrio cholerae were used for experiments after low temperature treatment. The samples were assessed by real-time and droplet digital PCR separately before and after low temperature treatment. Real-time fluorescence PCR experimental data were analyzed using the ??CT and geNorm methods. Droplet digital PCR experimental data were analyzed using the relative and absolute quantification methods to calculate the fold change in gene expression in the treatment group as compared to the control group. The data from the four gene quantification methods were analyzed using the multidimensional scaling method. Under these experimental conditions, the results indicated that there was a significant difference between the results of the four gene quantification methods. The expression level of the 16S rRNA gene changed, making it unsuitable as a reference gene. Droplet digital PCR could give more intuitive results regarding the amount of change in gene expression and have better accuracy in the relative quantitative analysis of differentially expressed genes.
