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Int. J. Mol. Sci. 2014, 15(12), 22155-22172; doi:10.3390/ijms151222155

Identification and Evaluation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis in Tea Plant (Camellia sinensis (L.) O. Kuntze)

1
College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China
2
USDA-Agricultural Research Service, Biosciences Research Lab, Sunflower and Plant Biology Research Unit, 1605 Albrecht Blvd N, Fargo, ND 58102, USA
3
Tea Research Institute, Chinese Academy of Agricultural Sciences, No. 9 South Meiling Road, Hangzhou 310008, Zhejiang, China
4
National Center for Tea Improvement, Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture, No. 9 South Meiling Road, Hangzhou 310008, Zhejiang, China
*
Author to whom correspondence should be addressed.
Received: 12 October 2014 / Revised: 24 November 2014 / Accepted: 24 November 2014 / Published: 2 December 2014
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Abstract

Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a crucial step in qRT-PCR normalization. To date, only a few housekeeping genes have been identified and used as reference genes in tea plant. The validity of those reference genes are not clear since their expression stabilities have not been rigorously examined. To identify more appropriate reference genes for qRT-PCR studies on tea plant, we examined the expression stability of 11 candidate reference genes from three different sources: the orthologs of Arabidopsis traditional reference genes and stably expressed genes identified from whole-genome GeneChip studies, together with three housekeeping gene commonly used in tea plant research. We evaluated the transcript levels of these genes in 94 experimental samples. The expression stabilities of these 11 genes were ranked using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ∆CT method. Results showed that the three commonly used housekeeping genes of CsTUBULIN1, CsACINT1 and Cs18S rRNA1 together with CsUBQ1 were the most unstable genes in all sample ranking order. However, CsPTB1, CsEF1, CsSAND1, CsCLATHRIN1 and CsUBC1 were the top five appropriate reference genes for qRT-PCR analysis in complex experimental conditions. View Full-Text
Keywords: Camellia sinensis; reference gene; qRT-PCR; tea plant; gene expression; normalization Camellia sinensis; reference gene; qRT-PCR; tea plant; gene expression; normalization
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Hao, X.; Horvath, D.P.; Chao, W.S.; Yang, Y.; Wang, X.; Xiao, B. Identification and Evaluation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis in Tea Plant (Camellia sinensis (L.) O. Kuntze). Int. J. Mol. Sci. 2014, 15, 22155-22172.

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