Validation of reference genes aiming accurate normalization of qRT-PCR data in Dendrocalamus latiflorus Munro.
BACKGROUND
Dendrocalamus latiflorus Munro distributes widely in subtropical areas and plays vital roles as valuable natural resources. The transcriptome sequencing for D. latiflorus Munro has been performed and numerous genes especially those predicted to be unique to D. latiflorus Munro were revealed. qRT-PCR has become a feasible approach to uncover gene expression profiling, and the accuracy and reliability of the results obtained depends upon the proper selection of stable reference genes for accurate normalization. Therefore, a set of suitable internal controls should be validated for D. latiflorus Munro.


RESULTS
In this report, twelve candidate reference genes were selected and the assessment of gene expression stability was performed in ten tissue samples and four leaf samples from seedlings and anther-regenerated plants of different ploidy. The PCR amplification efficiency was estimated, and the candidate genes were ranked according to their expression stability using three software packages: geNorm, NormFinder and Bestkeeper. GAPDH and EF1α were characterized to be the most stable genes among different tissues or in all the sample pools, while CYP showed low expression stability. RPL3 had the optimal performance among four leaf samples. The application of verified reference genes was illustrated by analyzing ferritin and laccase expression profiles among different experimental sets. The analysis revealed the biological variation in ferritin and laccase transcript expression among the tissues studied and the individual plants.


CONCLUSIONS
geNorm, NormFinder, and BestKeeper analyses recommended different suitable reference gene(s) for normalization according to the experimental sets. GAPDH and EF1α had the highest expression stability across different tissues and RPL3 for the other sample set. This study emphasizes the importance of validating superior reference genes for qRT-PCR analysis to accurately normalize gene expression of D. latiflorus Munro.
State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing, People's Republic of China ; The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, People's Republic of China.
Mingying
M
Liu
State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing, People's Republic of China ; The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, People's Republic of China.
Jing
J
Jiang
State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing, People's Republic of China ; The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, People's Republic of China.
Xiaojiao
X
Han
State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing, People's Republic of China ; The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, People's Republic of China.
Guirong
G
Qiao
State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing, People's Republic of China ; The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, People's Republic of China.
Renying
R
Zhuo
D019295:Computational Biology / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D006109:Poaceae / Q000235:genetics*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins / Q000235:genetics; D012984:Software
D020648:Peptide Elongation Factor 1; D010940:Plant Proteins; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases