Evaluation of Appropriate Reference Genes for Reverse Transcription-Quantitative PCR Studies in Different Tissues of a Desert Poplar via Comparision of Different Algorithms.
Despite the unshakable status of reverse transcription-quantitative PCR in gene expression analysis, it has certain disadvantages, including that the results are highly dependent on the reference genes selected for data normalization. Since inappropriate endogenous control genes will lead to inaccurate target gene expression profiles, the validation of suitable internal reference genes is essential. Given the increasing interest in functional genes and genomics of Populus euphratica, a desert poplar showing extraordinary adaptation to salt stress, we evaluated the expression stability of ten candidate reference genes in P. euphratica roots, stems, and leaves under salt stress conditions. We used five algorithms, namely, ΔCt, NormFinder, geNorm, GrayNorm, and a rank aggregation method (RankAggreg) to identify suitable normalizers. To support the suitability of the identified reference genes and to compare the relative merits of these different algorithms, we analyzed and compared the relative expression levels of nine P. euphratica functional genes in different tissues. Our results indicate that a combination of multiple reference genes recommended by GrayNorm algorithm (e.g., a combination of Actin, EF1α, GAPDH, RP, UBQ in root) should be used instead of a single reference gene. These results are valuable for research of gene identification in different P. euphratica tissues.
National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. whling@bjfu.edu.cn.|The Key Laboratory for Silviculture and Conservation of Ministry Education, College of Forestry, Beijing Forestry University, Beijing 100083, China. whling@bjfu.edu.cn.
Hou-Ling
HL
Wang
National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. lilan@bjfu.edu.cn.
Lan
L
Li
Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China. tangsha@caas.cn.
Sha
S
Tang
National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. yuanchao@bjfu.edu.cn.
Chao
C
Yuan
National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. tqqetqq@bjfu.edu.cn.
Qianqian
Q
Tian
National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. yanyansu@bjfu.edu.cn.
Yanyan
Y
Su
National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. hg_li@bjfu.edu.cn.
Hui-Guang
HG
Li
National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. lynnzhao@bjfu.edu.cn.
Lin
L
Zhao
National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. yinwl@bjfu.edu.cn.|The Key Laboratory for Silviculture and Conservation of Ministry Education, College of Forestry, Beijing Forestry University, Beijing 100083, China. yinwl@bjfu.edu.cn.
Weilun
W
Yin
National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. ruizhao@bjfu.edu.cn.
Rui
R
Zhao
National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. xiaxl@bjfu.edu.cn.
Xinli
X
Xia
D000465:Algorithms; D019295:Computational Biology / Q000379:methods*; D003889:Desert Climate; D020869:Gene Expression Profiling / Q000379:methods; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D009928:Organ Specificity / Q000235:genetics; D032107:Populus / Q000235:genetics*; D020871:RNA Stability; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013312:Stress, Physiological / Q000235:genetics; D059467:Transcriptome
Populus euphratica; normalization; reference genes; reverse transcription-quantitative PCR; salt stress