Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze)

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.

Figure 1

Average cycle threshold (C_T) values for 11 candidate reference genes. The filled diamond symbol indicates the mean of C_T values. The bars indicate standard deviation.

Figure 2

Expression profile of candidate reference genes and target genes under different experimental conditions. Six experimental series including 94 samples were used to examine gene expression. The fold difference is designated as log2 value. Red indicates up-regulated genes and green indicates down-regulated genes as compared with first sampling point/tissue. Bars at the bottom indicate the range of transcript changes in log2 value. The range of transcript changes for each experimental series is provided inside the parenthesis.