title: Selection and validation of reference genes for real-time quantitative PCR in hyperaccumulating ecotype of Sedum alfredii under different heavy metals stresses.
abstract: Real-time Quantitative PCR (RT-qPCR) has become an effective method for accurate analysis of gene expression in several biological systems as well as under different experimental conditions. Although with high sensitivity, specificity and broad dynamic range, this method requires suitable reference genes for transcript normalization in order to guarantee reproducible and meaningful results. In the present study, we evaluated five traditional housekeeping genes and five novel reference genes in Hyperaccumulating ecotype of Sedum alfredii, a well known hyperaccumulator for heavy metals phytoremediation, under Cd, Pb, Zn and Cu stresses of seven different durations. The expression stability of these ten candidates were determined with three programs--geNorm, NormFinder and BestKeeper. The results showed that all the selected reference genes except for SAND could be used for RT-qPCR normalization. Among them UBC9 and TUB were ranked as the most stable candidates across all samples by three programs together. For the least stable reference genes, however, BestKeeper produced different results compared with geNorm and NormFinder. Meanwhile, the expression profiles of PCS under Cd, Pb, Zn and Cu stresses were assessed using UBC9 and TUB respectively, and similar trends were obtained from the results of the two groups. The distinct expression patterns of PCS indicated that various strategies could be taken by plants in adaption to different heavy metals stresses. This study will provide appropriate reference genes for further gene expression quantification using RT-qPCR in Hyperaccumulator S. alfredii.
authors: State Key Laboratory of Tree Genetics and Breeding, Beijing, China ; Key Laboratory of Tree Genomics, The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, Zhejiang, China.
authors: Jian
authors: J
authors: Sang
authors: Xiaojiao
authors: X
authors: Han
authors: Mingying
authors: M
authors: Liu
authors: Guirong
authors: G
authors: Qiao
authors: Jing
authors: J
authors: Jiang
authors: Renying
authors: R
authors: Zhuo
mesh_terms: D000465:Algorithms; D001483:Base Sequence; D001673:Biodegradation, Environmental; D002104:Cadmium / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D060146:Ecotype; D020869:Gene Expression Profiling / Q000379:methods*; D018506:Gene Expression Regulation, Plant; D020043:Genes, Essential; D017343:Genes, Plant; D007854:Lead / Q000737:chemistry; D019216:Metals, Heavy / Q000737:chemistry*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D031963:Sedum / Q000187:drug effects / Q000235:genetics*; D015032:Zinc / Q000737:chemistry
chemical_list: D017931:DNA Primers; D019216:Metals, Heavy; D010940:Plant Proteins; D002104:Cadmium; D007854:Lead; D003300:Copper; D015032:Zinc