Genome-wide selection of superior reference genes for expression studies in Ganoderma lucidum.
Quantitative real-time polymerase chain reaction (qRT-PCR) is widely used for the accurate analysis of gene expression. However, high homology among gene families might result in unsuitability of reference genes, which leads to the inaccuracy of qRT-PCR analysis. The release of the Ganoderma lucidum genome has triggered numerous studies to be done on the homology among gene families with the purpose of selecting reliable reference genes. Based on the G. lucdum genome and transcriptome database, 38 candidate reference genes including 28 novel genes were systematically selected and evaluated for qRT-PCR normalization. The result indicated that commonly used polyubiquitin (PUB), beta-actin (BAT), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were unsuitable reference genes because of the high sequence similarity and low primer specificity. According to the evaluation of RefFinder, cyclophilin 5 (CYP5) was ranked as the most stable reference gene for 27 tested samples under all experimental conditions and eighteen mycelial samples. Based on sequence analysis and expression analysis, our study suggested that gene characteristic, primer specificity of high homologous genes, allele-specificity expression of candidate genes and under-evaluation of reference genes influenced the accuracy and sensitivity of qRT-PCR analysis. This investigation not only revealed potential factors influencing the unsuitability of reference genes but also selected the superior reference genes from more candidate genes and testing samples than those used in the previous study. Furthermore, our study established a model for reference gene analysis by using the genomic sequence.
The National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China. Electronic address: xuzhichao@implad.ac.cn.
Zhichao
Z
Xu
Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Science, Beijing 100700, China. Electronic address: xj8442cn@126.com.
Jiang
J
Xu
The National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China. Electronic address: ajli@implad.ac.cn.
Aijia
A
Ji
Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Science, Beijing 100700, China. Electronic address: yjzhu@implad.ac.cn.
Yingjie
Y
Zhu
The National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China. Electronic address: zhangxin@implad.ac.cn.
Xin
X
Zhang
School of Life Sciences, Peking University, Beijing 100871, China. Electronic address: huyl@pku.edu.cn.
Yuanlei
Y
Hu
The National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China; Chongqing Institute of Medicinal Plant Cultivation, Chongqing 408435, China. Electronic address: jysong@implad.ac.cn.
Jingyuan
J
Song
The National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China; Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Science, Beijing 100700, China. Electronic address: slchen@implad.ac.cn.
Shilin
S
Chen
D000199:Actins / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D018506:Gene Expression Regulation, Plant; D020043:Genes, Essential; D017343:Genes, Plant; D056726:Genetic Association Studies / Q000592:standards; D018745:Genome, Plant; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D025821:Polyubiquitin / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012015:Reference Standards; D020940:Reishi / Q000235:genetics*; D059467:Transcriptome
D000199:Actins; D017931:DNA Primers; D025821:Polyubiquitin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
BAT; CYP5; Ganoderma lucidum; High homologous genes; PUB; qRT-PCR