Evaluation and validation of internal control genes for studying gene expression in the dinoflagellate Prorocentrum minimum using real-time PCR

Housekeeping genes (HKGs) are required for the normalization of expression levels in real-time PCR, and their selection is critical for gene expression studies. However, stable expressions of candidate HKGs vary among organisms and tissues and even according to environmental conditions. Here, we evaluated the gene expressions of 10 frequently used HKGs, including 18S rRNA, P2, ACT, TUA, TUB, CYC, eIF4E, MDH, UBQ, and GAPDH, with a total of 17 RNA samples extracted from the dinoflagellate Prorocentrum minimum. All the RNAs were prepared from various cells cultured under different conditions, including thermal shocks, toxic chemical exposures, and different life stages. Via C(T) analyses of the 10 HKGs using the geNorm software, TUA was selected as the most stable HKG for gene expression studies of the dinoflagellate, followed by MDH. Pair-wise variation (V) analysis showed that at least 2 genes were required for accurate normalization of gene expression studies depending on the experimental conditions. These HKGs are useful internal controls for the normalization of gene expression in P. minimum.     

The glyceraldehyde-3-phosphate dehydrogenase gene of Moniliophthoraperniciosa, the causal agent of witches' broom disease of Theobroma cacao

This report describes the cloning, sequence and expression analysis of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of Moniliophthora perniciosa, the most important pathogen of cocoa in Brazil. Southern blot analysis revealed the presence of a single copy of the GAPDH gene in the M. perniciosa genome (MpGAPDH). The complete MpGAPDH coding sequence contained 1,461 bp with eight introns that were conserved in the GAPDH genes of other basidiomycete species. The cis-elements in the promoter region of the MpGAPDH gene were similar to those of other basidiomycetes. Likewise, the MpGAPDH gene encoded a putative 339 amino acid protein that shared significant sequence similarity with other GAPDH proteins in fungi, plants, and metazoans. Phylogenetic analyses clustered the MPGAPDH protein with other homobasidiomycete fungi of the family Tricholomataceae. Expression analysis of the MpGAPDH gene by real-time PCR showed that this gene was more expressed (~1.3X) in the saprotrophic stage of this hemibiotrophic plant pathogen than in the biotrophic stage when grown in cacao extracts.     

Evaluation of reference genes for real-time quantitative PCR studies in

ABSTRACT: BACKGROUND: 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. RESULTS: 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, EF1a, 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-[increment][increment]CT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-[increment][increment]CT 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. CONCLUSIONS: 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.     

桉蝙蛾幼虫实时荧光定量PCR内参基因的筛选

本研究通过筛选桉蝙蛾Endoclita signifer Walker幼虫不同龄期与不同体节中稳定表达的内参基因,为桉蝙蛾基因表达研究提供参考。通过实时荧光定量PCR技术测定5个候选内参基因（ACTIN、GAPDH、TUB、RIB、EF）在桉蝙蛾幼虫不同龄期（3龄、5龄、9龄、12龄）与不同体节（头部、胸部、腹部）及全样品（由所有样品组成）处理中的表达量,后利用GeNorm、NormFinder、BestKeeper对5个候选基因的稳定性进行评估,最后由RefFinder综合分析结果,选出最佳的内参基因。基于4种分析方法的评估可知,桉蝙蛾5龄和9龄幼虫的不同体节、不同龄期幼虫的头部和胸部中内参基因的最佳数目为2,而3龄和12龄幼虫的不同体节、不同龄期幼虫的腹部及全样本中内参基因的最佳数目为3。3龄、5龄、9龄和12龄幼虫的不同体节中可分别选择ACTIN+RIB+GAPDH、EF+RIB、GAPDH+EF和ACTIN+RIB+GAPDH作为最稳定的内参基因组合;EF+RIB、RIB+GAPDH和EF+GAPDH+ACTIN可分别作为不同龄期幼虫头部、胸部和腹部的最佳内参基因组合;综合考虑桉蝙蛾幼虫不同龄期与不同体节的影响时,可选择RIB、ACTIN和GAPDH作为内参基因。     

实时荧光定量PCR分析中毛果杨内参基因的筛选和验证

实时荧光定量PCR(qRT-PCR)技术具有高灵敏性、高保真性和高特异性, 被广泛应用于基因表达的分析。在数据处理过程中, 选用稳定表达的基因作为内参基因对准确分析实验结果非常关键。以毛果杨(Populus trichocarpa)的不同组织以及锌胁迫下的组培苗为材料, 使用荧光定量PCR方法分析了TUA8、TUB6、ubiquitin、GAPDH、actin、18S rRNA和EF1α 7个看家基因的表达情况。通过geNorm、NormFinder和BestKeeper 3个程序的综合分析, 发现actin、ubiquitin、EF1α和18S rRNA的稳定性较好, 可用作毛果杨基因表达研究的内参基因; 而TUB6在不同组织中稳定性最差; GAPDH在锌胁迫下的组织中稳定性最差, 因此不适宜作为内参基因。毛果杨NAC基因的表达分析, 进一步验证了上述结果。该研究对采用qRT-PCR方法分析毛果杨基因表达过程中内参基因的选择具有指导作用, 同时对揭示NAC基因的功能也有一定的意义。     

Selection and Evaluation of Reference Genes for Expression Analysis Using qRT-PCR in the Beet Armyworm Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae)

Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring and evaluating changes in gene expression. The most common method for analyzing qRT-PCR data is to normalize mRNA levels of target genes to internal reference genes. Evaluating and selecting stable reference genes on a case-by-case basis is critical. The present study aimed to facilitate gene expression studies by identifying the most suitable reference genes for normalization of mRNA expression in qRT-PCR analysis of the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). For this purpose, three software tools (geNorm, NormFinder and BestKeeper) were used to investigate 10 candidate reference genes in nine developmental stages and five different tissues (epidermis, head, midgut, fat body and hemolymph) in three larval physiological stages (molting, feeding and wandering stages) of, S. exigua. With the exception of 18S ribosomal RNA (18S), all other candidate genes evaluated, β-actin1(ACT1), β-actin2 (ACT2), elongation factor1(EF1), elongation factor 2 (EF2), Glyceralde hyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (L10), ribosomal protein L17A (L17A), superoxide dismutase (SOD), α-tubulin (TUB),proved to be acceptable reference genes. However, their suitability partly differed between physiological stages and different tissues. L10, EF2 and L17A ranked highest in all tissue sample sets. SOD, ACT2, GAPDH, EF1 and ACT1 were stably expressed in all developmental stage sample sets; ACT2, ACT1 and L10 for larvae sample sets; GAPDH, ACT1 and ACT2 for pupae and adults; SOD and L17A for males; and EF2 and SOD for females. The expression stability of genes varied in different conditions. The findings provided here demonstrated, with a few exceptions, the suitability of most of the 10 reference genes tested in tissues and life developmental stages. Overall, this study emphasizes the importance of validating reference genes for qRT-PCR analysis in S. exigua.     

Validation of Zebrafish （Danio redo） Reference Genes for Quantitative Real-time RT-PCR Normalization

The normalization of quantitative real time RT-PCR （qRT-PCR） is important to obtain accurate gene expression data. The most common method for qRT-PCR normalization is to use reference, or housekeeping genes. However, there is emerging evidence that even reference genes can be regulated under different conditions, qRT-PCR has only recently been used in terms of zebrafish gene expression studies and there is no validated set of reference genes. This study characterizes the expression of nine possible reference genes during zebrafish embryonic development and in a zebrafish tissue panel. All nine reference genes exhibited variable expression. The fl-actin, EFlot and Rpll3ot genes comprise a validated reference gene panel for zebrafish developmental time course studies, and the EF1 or, Rpll3α and 18S rRNA genes are more suitable as a reference gene panel for zebrafish tissue analysis. Importantly, the zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies.     

Bt毒素诱导下小菜蛾实时定量PCR 内参基因的筛选

【目的】筛选出Bt毒素诱导后的小菜蛾Plutella xylostella (L.)的实时定量PCR最适内参基因。【方法】选取核糖体18S rRNA (18S rRNA)、 肌动蛋白（ACTB）、 延伸因子（EF1）、3-磷酸甘油醛脱氢酶（GAPDH）、 核糖体蛋白L32 (RPL32)、 核糖体蛋白S13 （RPS13）、 核糖体蛋白S20 （RPS20）和β-微管蛋白(TUB)基因作为候选内参基因, 以geNorm、 Normfinder和BestKeeper软件分析这8个基因在Bt毒素诱导后的小菜蛾不同品系中肠组织中的表达稳定性。并应用筛选出来的内参基因分析小菜蛾氨肽酶2（aminopeptidase N2, APN2）基因的表达水平。【结果】geNorm软件以RPS13和EF1为最稳定内参基因, NormFinder和BestKeeper软件均以RPS13和RPL32为最稳定基因。使用3种不同内参基因分析Bt毒素诱导后的小菜蛾Bt抗性和敏感品系中ANP2表达水平时, 新的内参基因EF1和传统内参基因RPL32表现了良好的稳定性, 二者作为标准化因子, ANP2表达量结果基本一致, 而使用18S rRNA作为内参基因, 却导致部分表达量分析结果有所误差。【结论】筛选出PRS13,RPL32和EF1可以作为小菜蛾某些试验条件下的内参基因, 对小菜蛾基因表达研究奠定了一定基础, 也对其他昆虫内参基因的筛选具有参考价值。     

冬虫夏草菌实时荧光定量PCR内参基因的筛选

以冬虫夏草单子囊孢子分离得到的菌株TZ8-1的3种菌丝形态为实验材料,提取RNA,经反转录获取cDNA,选择了 11个持家基因为候选内参基因(18SrRNA、APRTase、β-TUB、RPL2、EF1-α、PGI、PGM、H+-ATPase、ACT1、UBQ和GAPDH),根据该菌基因组注释结果来设计引物,采用实时荧...     

Validation of reference genes of grass carp Ctenopharyngodon idellus for the normalization of quantitative real-time PCR

Expression of four reference genes of grass carp, including β-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA (18S) and elongation factor-1 alpha (EF1α), was studied in tissues of normal individuals and bacteria-infected individuals. EF1α had the most stable expressions followed by 18S rRNA then GAPDH; ACTB had the least stability. After being infected with bacteria, the grass carp showed minimal changes in expression levels of EF1α in the liver and head kidney, while ACTB had the most stable expressions in spleen but the least stable in liver. EF1α is thus the optimal reference gene in quantitative real-time PCR analysis to quantitate the expression levels of target genes in tissues of grass carp.     

Evaluation of reference genes for real-time PCR studies of Brazilian Somalis sheep infected by gastrointestinal nematodes

Precise normalization with reference genes is necessary, in order to obtain reliable relative expression data in response to gastrointestinal nematode infection. By using sheep from temperate regions as models, three reference genes, viz., ribosomal protein LO (RPLO), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and succinate dehydrogenase complex subunit A (SDHA), were investigated in the abomasum, abomasal lymph nodes and small intestine of Brazilian Somalis sheep, either resistant or susceptible to gastrointestinal nematodes infections. Real time PCR was carried out by using SYBR Green I dye, and gene stability was tested by geNorm. RPLO was an ideal reference gene, since its expression was constant across treatments, presented lower variation, and was ranked as the most stable in abomasum and lymph node tissues. On the other hand, SDHA was the most stable in the small intestine followed by RPLO and GAPDH. These findings demonstrate the importance of correctly choosing reference genes prior to relative quantification. In addition, we determined that reference genes used in sheep from temperate regions, when properly tested, can be applied in animals from tropical regions such as the Brazilian Somalis sheep.     

夜香树花期荧光定量PCR内参基因的筛选

为筛选夜香树（Cestrum nocturnum L.）香气释放、生物钟等相关基因表达研究适用的内参基因,本研究采用夜香树盛花期叶片和花为实验材料,利用同源克隆和RACE技术,获得了夜香树6种经典的内参基因序列,分别为：Actb7、EF-1A、GAPDH、TUA、TUB2、UBQ;采用荧光定量PCR方法对18s rRNA和这6个内参基因的表达模式进行了分析,并通过Bestkeeper、geNorm、NormFinder 3种程序分析了内参基因的稳定性。结果表明,在花中,Actb7表达最稳定;在叶片中,EF-1A和UBQ的表达比较稳定;在2种组织中,EF-1A的表达相对稳定。3组稳定性分析中,geNorm程序确定的最佳内参基因数目均为2,最佳内参基因组合均为Actb7/EF-1A。本研究通过对稳定内参基因的筛选,以期为准确检测夜香树盛花期花瓣节律运动、香气释放、生物钟变化等相关基因的表达研究奠定基础。     

Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress

We examined eight putative consistently expressed genes—actin (ACT), β-tubulin, elongation factor 1α (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), ribosomal protein L2 (RPL2), ubiquitin (UBI), and a catalytic subunit of protein phosphatase 2A (PP2Acs)—for their potential as references for the normalization of gene expression in tomato leaves. Expression stability of candidate reference genes was tested during growth conditions of nitrogen (N) starvation, low temperature, and suboptimal light. The geNorm algorithm, using reciprocal cross-validation among a larger group of candidate references, was applied for this purpose. The widely used reference genes GAPDH and PGK were top ranked during light stress but poorly ranked during N and cold stress. In contrast, EF1 was top ranked during N and cold stress but poorly ranked during light stress. The novel references RPL2 and PP2Acs, as well as the traditional references ACT and UBI, appeared to be stably expressed when looking at the data set as a whole. No gene was identified that exhibited such a constant level of expression as to outperform the other candidates under all experimental conditions. Thus, the results highlight the need for normalizing gene expression in tomato using the geometric average of multiple carefully selected reference genes.