Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment.
Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment.
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DOI：
10.1186/1471-2199-13-22
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发表时间：
2012-06-29
影响因子：
--
通讯作者：
Bennett JE
中科院分区：
文献类型：
--
作者：
Li QQ;Skinner J;Bennett JE
实时荧光定量PCR（real-time quantitative PCR，RT-qPCR）参考基因的选择是在不同实验条件下进行可靠基因表达分析的重要前提。本研究旨在通过RT-qPCR在唑刺激的光滑念珠菌中鉴定作为基因表达研究的内部对照的参考基因。使用hkgestion工具计算倍数变化和标准差，评估16个参考基因在氟康唑胁迫下的表达稳定性。我们的数据显示，三种核糖体RNA（RDN5.8，RDN 18和RDN 25）的mRNA表达水平在氟康唑的作用下保持稳定，而PGK 1，UBC 7和UBC 13 mRNA分别仅显示约2.9，3.0和2.5倍的唑诱导。与此相反，其他10个参考基因（ACT 1、EF 1 α、GAPDH、PPIA、RPL 2A、RPL 10、RPL 13 A、SDHA、TUB 1和UBC 4）的mRNA水平在C.光滑的抗真菌治疗后，表现出的变化范围为4.5- 32.7倍。我们还使用2-ΔΔCT方法和其他三个软件包评估了这些参考基因的表达稳定性。geNorm法和2-ΔΔCT法对参考基因的稳定性排序结果与hkgal法一致，而BestKeeper法和Normalt法对参考基因的稳定性排序结果差异显著。然后，我们使用比较CT方法验证了六个候选参考基因（ACT 1，PGK 1，RDN5.8，RDN 18，UBC 7和UBC 13）作为该系统中十个靶基因的内部对照的适用性。我们的验证实验通过了所有六个参考基因分析，除了RDN 18，其中RDN 18的扩增效率与十个靶基因的扩增效率不同。最后，我们证明了靶基因表达的相对定量根据所使用的内源性对照而变化，突出了在此类实验中选择内部对照的重要性。我们建议使用RDN5.8、UBC 13和PGK 1单独或RDN5.8 + UBC 13或PGK 1的组合作为参考基因用于C.唑类药物治疗后出现光滑。相反，我们发现ACT 1和其他常用的参考基因（GAPDH，PPIA，RPL 13 A，TUB 1等）。在当前模型中未被验证为良好的内部控制。
The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata. The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25) remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1α, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4) were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2-ΔΔCT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-ΔΔCT method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13) as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such experiments. We recommend the use of RDN5.8, UBC13, and PGK1 alone or the combination of RDN5.8 plus UBC13 or PGK1 as reference genes for RT-qPCR analysis of gene expression in C. glabrata following azole treatment. In contrast, we show that ACT1 and other commonly used reference genes (GAPDH, PPIA, RPL13A, TUB1, etc.) were not validated as good internal controls in the current model.
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影响因子：
3.7
作者：
de Jonge HJ;Fehrmann RS;de Bont ES;Hofstra RM;Gerbens F;Kamps WA;de Vries EG;van der Zee AG;te Meerman GJ;ter Elst A
通讯作者：
ter Elst A
DOI：
10.1016/s0169-328x(02)00417-5
发表时间：
2002-10-15
期刊：
MOLECULAR BRAIN RESEARCH
影响因子：
--
作者：
Bond, BC;Virley, DJ;Preece, P
通讯作者：
Preece, P
影响因子：
2.7
作者：
Dheda, K;Huggett, JF;Zumla, A
通讯作者：
Zumla, A
影响因子：
2.7
作者：
Abruzzo, LV;Lee, KY;Coombes, KR
通讯作者：
Coombes, KR
影响因子：
2.9
作者：
BHATIA, P;TAYLOR, WR;WRIGHT, JA
通讯作者：
WRIGHT, JA