TY - JOUR
T1 - Genome-wide selection of superior reference genes for expression studies in Ganoderma lucidum
AU - Xu, Zhichao
AU - Xu, Jiang
AU - Ji, Aijia
AU - Zhu, Yingjie
AU - Zhang, Xin
AU - Hu, Yuanlei
AU - Song, Jingyuan
AU - Chen, Shilin
N1 - Publisher Copyright:
© 2015 Elsevier B.V.
PY - 2015/12/15
Y1 - 2015/12/15
N2 - 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.
AB - 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.
KW - BAT
KW - CYP5
KW - Ganoderma lucidum
KW - High homologous genes
KW - PUB
KW - QRT-PCR
UR - http://www.scopus.com/inward/record.url?scp=84946476561&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84946476561&partnerID=8YFLogxK
U2 - 10.1016/j.gene.2015.08.025
DO - 10.1016/j.gene.2015.08.025
M3 - Article
C2 - 26277249
AN - SCOPUS:84946476561
SN - 0378-1119
VL - 574
SP - 352
EP - 358
JO - Gene
JF - Gene
IS - 2
M1 - 40772
ER -