Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze).
Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze).
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DOI：
10.3390/ijms151222155
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发表时间：
2014-12-02
影响因子：
5.6
通讯作者：
Xiao B
中科院分区：
文献类型：
--
作者：
Hao X;Horvath DP;Chao WS;Yang Y;Wang X;Xiao B
在各种实验条件下准确定量基因表达的可靠参考选择是qRT-PCR标准化的关键步骤。迄今为止，只有少数管家基因已被鉴定并用作茶树的参考基因。这些参考基因的有效性尚不清楚，因为它们的表达稳定性尚未得到严格的检验。为了筛选更适合茶树qRT-PCR研究的参考基因，我们检测了来自3个不同来源的11个候选参考基因的表达稳定性：拟南芥传统参考基因的直向同源物、全基因组基因芯片研究中鉴定的稳定表达基因，以及茶树研究中常用的3个管家基因。我们评估了94个实验样本中这些基因的转录水平。这11个基因的表达稳定性排名使用四种不同的计算程序，包括geNorm，Normfinder，BestKeeper，和比较的RT-PCR方法。结果表明，CsTUBULIN 1、CsACINT 1、Cs 18 S rRNA 1和CsUBQ 1这3个常用的管家基因在所有样本排序中均为最不稳定的基因。然而，CsPTB 1、CsEF 1、CsSAND 1、CsCLATHRIN 1和CsUBC 1是在复杂实验条件下进行qRT-PCR分析的前5个合适的参考基因。
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.
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影响因子：
5.3
作者：
Artico S;Nardeli SM;Brilhante O;Grossi-de-Sa MF;Alves-Ferreira M
通讯作者：
Alves-Ferreira M
影响因子：
2.7
作者：
Dheda, K;Huggett, JF;Zumla, A
通讯作者：
Zumla, A
影响因子：
3.1
作者：
Migocka, M.;Papierniak, A.
通讯作者：
Papierniak, A.
影响因子：
4.9
作者：
IINO, M
通讯作者：
IINO, M
影响因子：
10.5
作者：
Lee, Jeong Hwan;Yoo, Seong Jeon;Ahn, Ji Hoon
通讯作者：
Ahn, Ji Hoon