Reference genes for quantitative analysis on Clonorchis sinensis gene expression by real-time PCR.
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 from Clonorchis sinensis metacercariae 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, beta-actin, and calcyphosine; reference genes found to be stably expressed under gamma-irradiated and non-irradiated conditions were succinate dehydrogenase, small nuclear ribonucleoprotein, and beta-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 the C (T) values of single reference genes and the geometric mean of the C (T) values of three reference genes. When comparing C. sinensis gene expressions, we propose to employ the geometric mean of the C (T) values of more than three reference genes validated in the same experimental setting.
Yoo
Won Gi
WG
Department of Medical Environmental Biology, Chung-Ang University College of Medicine, Tongjak-gu, Seoul 156-756, Republic of Korea.
Kim
Tae Im
TI
Li
Shunyu
S
Kwon
Oh Sil
OS
Cho
Pyo Yun
PY
Kim
Tong-Soo
TS
Kim
Kijeong
K
Hong
Sung-Jong
SJ
D000818:Animals; D003004:Clonorchis sinensis / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012015:Reference Standards