四种基因定量方法对实时荧光PCR与微滴式数字PCR检测霍乱弧菌基因表达量的分析

Real-time and Droplet Digital PCR Analyses for Detection of Vibrio cholerae Gene Expression by Four Gene Quantification Methods

摘要:

基因表达量测定对基因功能解析和探索生命机理具有重大意义，目前实时荧光PCR和微滴式数字PCR技术是进行基因表达量测定的有效方法。本文以低温处理的霍乱弧菌sfs、vcc、RecA、hly及16S rRNA基因为研究对象，分别用实时荧光PCR和微滴式数字PCR技术对低温处理前后霍乱弧菌的上述基因表达量进行测定，实时荧光PCR实验数据分别采用??CT和geNorm法进行分析，微滴式数字PCR实验数据分别采用相对定量和绝对定量法进行分析，获得处理组基因表达量相对于对照组的变化倍数；使用多维尺度法来分析四种基因定量方法的数据。结果表明，在本实验条件下，四种基因定量方法的分析结果存在差异；16S rRNA基因表达量发生了变化，不适合作为内参基因；微滴式数字PCR能更直观的给出基因表达量变化的结果，并且基因表达差异相对定量分析的准确度更高。

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.