Reference genes for quantitative analysis on Clonorchis sinensis gene expression by real-time PCR
Reference genes for quantitative analysis on Clonorchis sinensis gene expression by real-time PCR
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DOI：
10.1007/s00436-008-1195-x
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发表时间：
2008
影响因子：
2
通讯作者：
W. Yoo;Tae Im Kim;Shunyu Li;O. S. Kwon;P. Cho;Tong‐Soo Kim;Kijeong Kim;Sung-Jong Hong
中科院分区：
文献类型：
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作者：
W. Yoo;Tae Im Kim;Shunyu Li;O. S. Kwon;P. Cho;Tong‐Soo Kim;Kijeong Kim;Sung-Jong Hong
通过定量实时聚合酶链式反应测定的相对基因表达的准确性很大程度上取决于所使用的参考基因的变异性。验证实验条件下内参基因的稳定性是比较研究实验组中目的基因表达水平的重要初始步骤。使用从华支睾吸虫和成虫中独立提取的三个总RNA样本，我们使用geNorm程序确定了八个参考基因候选物的基因表达稳定性和三个目标基因的相对转录水平。发现在囊蚴和成虫中稳定表达的内参基因是磷酸甘油酸激酶、β-肌动蛋白和卡赛膦；在γ辐射和非辐射条件下稳定表达的参考基因是琥珀酸脱氢酶、小核核糖核蛋白和β-肌动蛋白；无论胆汁处理如何，稳定表达的是小核核糖核蛋白、磷酸甘油酸激酶和琥珀酸脱氢酶。根据我们的数据，目标基因的表达水平取决于归一化因素，例如单个参考基因的CT值和三个参考基因的CT值的几何平均值。比较时 C.对于sinensis基因表达，我们建议采用在相同实验环境中验证的三个以上参考基因的CT值的几何平均值。
The accuracies of relative gene expressions as determined by quantitative real-time polymerase chain reaction are largely dependent on the variabilities of the reference genes used. Validation of the stabilities of reference genes under experimental conditions is an essential initial step for comparative studies on the expression levels of target genes in experimental groups. Using three total RNA samples extracted independently fromClonorchis sinensismetacercariae and adults, we determined the gene expression stabilities of eight reference gene candidates and the relative transcript levels of three target genes using the geNorm program. The reference genes found to be stably expressed in metacercariae and adults were phosphoglycerate kinase, β-actin, and calcyphosine; reference genes found to be stably expressed under γ-irradiated and non-irradiated conditions were succinate dehydrogenase, small nuclear ribonucleoprotein, and β-actin; and those stably expressed regardless of bile treatment were small nuclear ribonucleoprotein, phosphoglycerate kinase, and succinate dehydrogenase. According to our data, the expression levels of target genes are dependent on normalization factors, such as theCTvalues of single reference genes and the geometric mean of theCTvalues of three reference genes. When comparingC. sinensisgene expressions, we propose to employ the geometric mean of theCTvalues of more than three reference genes validated in the same experimental setting.