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

Authors: HAO, XINYUAN - Northwest Agriculture And Forestry University; Horvath, David; Chao, Wun; YANG, YAJUN - Northwest Agriculture And Forestry University; WANG, XINCHAO - Tea Research Institute; XIAO, BIN - Northwest Agriculture And Forestry University

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/24/2014
Publication Date: 12/2/2014
Citation: Hao, X., Horvath, D.P., Chao, W.S., Yang, Y., Wang, X. and Xiao, B. 2014. Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze). International Journal of Molecular Sciences. 15:22155-22172. https://doi.org/10.3390/ijms151222155.
DOI: https://doi.org/10.3390/ijms151222155

Interpretive Summary: We note that many of the controls used to determine accumulation levels of specific RNAs in the agronomically very important crop tea plant (Camellia sinensis) may not accurately quantify the level of RNA. To remedy this situation for ourselves and others, we have examined the accumulation of various control genes from tea plant and identified a set of 5 suitable genes for use in normalizing the accumulation level of specific genes of interest to compensate for differences in the amount of total RNA that might be present in a given sample or for differences in the efficiency of amplification of any given gene in a sample. Additionally we determined that specific control genes worked better in some analyses than others. We expect our new data to be very useful for other researchers using qRT-PCR technology to generate accurate data.

Technical Abstract: Quantitative real-time polymerase chain reaction (qRT-PCR) is a commonly used technique for measuring gene expression levels due to its simplicity, specificity, and sensitivity. Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a crucial step in qRT-PCR normalization. So far, only a few housekeeping genes have been identified in tea plant, an emerging model system and important cash crop. To discover more appropriate reference genes for qRT-PCR studies on tea plant, we examined the stability of the RNA levels from tea plant homologues of traditional candidate reference genes from Arabidopsis (GAPDH, EF, UBQ and UBC) and stably expressed genes identified from whole-genome GeneChip studies (clathrin, SAND, TIP and PTB), together with three housekeeping gene currently used in tea plant studies (18S rRNA, actin, tubulin). We evaluated the transcript levels of these genes in 94 experimental samples consisting of diurnal variety (24 samples), different organs (18 samples), different duration of cold stress in leaves and shoots (18 samples for each), and different time points after auxin antagonist treatment (8 samples) and lanolin treatment (8 samples). The expression stabilities of these eleven genes were ranked using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative 'CT method. Results showed that the three housekeeping genes of tublin, actin and 18S rRNA together with UBQ were the most unstable genes in all sample ranking order. On the contrary, PTB, EF SAND, clathrin and UBC were the top five appropriate reference genes for qRT-PCR analysis in the complex experiential conditions. Specifically, EF was the most stable gene for diurnal expression; clathrin for different organs and different duration of cold and short day treatment in leaves; UBC for different duration of cold and short day treatment in shoots and lanolin treatment; PTB for auxin antagonist treatment.