Evaluation of Appropriate Reference Genes for Reverse Transcription-Quantitative PCR Studies in Different Tissues of a Desert Poplar via Comparision of Different Algorithms

Hou-Ling Wang; Lan Li; Sha Tang; Chao Yuan; Qianqian Tian; Yanyan Su; Hui-Guang Li; Lin Zhao; Weilun Yin; Rui Zhao; Xinli Xia

doi:10.3390/ijms160920468

Evaluation of Appropriate Reference Genes for Reverse Transcription-Quantitative PCR Studies in Different Tissues of a Desert Poplar via Comparision of Different Algorithms

Int J Mol Sci. 2015 Aug 28;16(9):20468-91. doi: 10.3390/ijms160920468.

Authors

Hou-Ling Wang^{1

2}, Lan Li³, Sha Tang⁴, Chao Yuan⁵, Qianqian Tian⁶, Yanyan Su⁷, Hui-Guang Li⁸, Lin Zhao⁹, Weilun Yin^{10

11}, Rui Zhao¹², Xinli Xia¹³

Affiliations

¹ National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. whling@bjfu.edu.cn.
² The Key Laboratory for Silviculture and Conservation of Ministry Education, College of Forestry, Beijing Forestry University, Beijing 100083, China. whling@bjfu.edu.cn.
³ National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. lilan@bjfu.edu.cn.
⁴ Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China. tangsha@caas.cn.
⁵ National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. yuanchao@bjfu.edu.cn.
⁶ National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. tqqetqq@bjfu.edu.cn.
⁷ National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. yanyansu@bjfu.edu.cn.
⁸ National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. hg_li@bjfu.edu.cn.
⁹ National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. lynnzhao@bjfu.edu.cn.
¹⁰ National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. yinwl@bjfu.edu.cn.
¹¹ The Key Laboratory for Silviculture and Conservation of Ministry Education, College of Forestry, Beijing Forestry University, Beijing 100083, China. yinwl@bjfu.edu.cn.
¹² National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. ruizhao@bjfu.edu.cn.
¹³ National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. xiaxl@bjfu.edu.cn.

Abstract

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.

Keywords: Populus euphratica; normalization; reference genes; reverse transcription-quantitative PCR; salt stress.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Algorithms*
Computational Biology / methods*
Desert Climate*
Gene Expression Profiling* / methods
Gene Expression Regulation, Plant*
Genes, Plant*
Organ Specificity / genetics
Populus / genetics*
RNA Stability
Reproducibility of Results
Sensitivity and Specificity
Stress, Physiological / genetics
Transcriptome