Selection of housekeeping genes for qRT-PCR analysis in potato tubers under cold stress

Quantitative real-time polymerase chain reaction (qRT-PCR) is currently the most sensitive method used for quantitative gene expression studies. However, minimal variation in the amount of material and presence of inhibitors affecting enzyme efficiency can lead to significant quantification errors. Accurate data normalization is vital using reference genes as internal controls. Many so-called housekeeping genes or reference genes with assumed stable expression can exhibit either up- or downregulation depending on the developmental stage or other environmental conditions. We have evaluated six reference genes (actin, APRT, 18S rRNA, ef1α, β-tubulin and ribosomal protein L2) for qRT-PCR profiling experiments in potato tuber tissues of five varieties during cold storage at different temperatures and treatment periods. Genes were ranked according to their expression stability by BestKeeper, geNorm and NormFinder software tools in the same order. This means that any of them can be used for this purpose. The results indicated that ef1α and APRT were the most stably expressed genes in the potato tuber tissues under different cold storage regimes. We therefore recommend use of this pair of genes as internal controls for gene expression studies under the described conditions.     

Selection and evaluation of reference genes for qRT-PCR of Scutellaria baicalensis Georgi under different experimental conditions

Molecular Biology Reports - Scutellaria baicalensis Georgi is a famous medicinal plant with its dried roots having been used as a traditional Chinese medicinal for more than 2000 years....     

Selection of reliable reference genes for qRT-PCR analysis in human non-cancerous gastric tissue

Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in complex diseases like obesity and gastritis. However, variations in amount of starting material, enzymatic efficiency and presence of amplification inhibitors can lead to quantification errors. Hence, the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Human gastric tissue has been the least investigated for stability of reference gene expression. In this study, three popular algorithms, GeNorm, NormFinder and BestKeeper were used to evaluate the reference gene stability. Conclusion: HPRT1 and GAPDH are the best performing pair of reference genes for qRT-PCR profiling experiments involving non-malignant gastric tissue samples.     

Selection of reliable reference genes for qRT-PCR analysis in human non-cancerous gastric tissue

Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in complex diseases like obesity and gastritis. However, variations in amount of starting material, enzymatic efficiency and presence of amplification inhibitors can lead to quantification errors. Hence, the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Human gastric tissue has been the least investigated for stability of reference gene expression. In this study, three popular algorithms, GeNorm, NormFinder and BestKeeper were used to evaluate the reference gene stability. Conclusion: HPRT1 and GAPDH are the best performing pair of reference genes for qRT-PCR profiling experiments involving non-malignant gastric tissue samples.     

Selection of suitable reference genes for quantitative real-time PCR in trabecular meshwork cells under oxidative stress

Oxidative stress-induced dysfunction in trabecular meshwork (TM) cells is considered a major alteration that can lead to glaucoma. Hydrogen peroxide (H₂O₂) is the most widely used agent for inducing oxidation in TM cells in vitro. Quantitative real-time PCR (qPCR) is an important method for studying alterations in gene expression, and suitable (i.e. invariant) reference genes must be defined to normalize expression levels. In this study, eight common reference genes, i.e. PRS18, ACTB, B2M, GAPDH, PPIA, HPRT1, YWHAZ, and TBP, were evaluated for use in studies of H₂O₂-induced dysfunction in TM cells. Three established algorithms, geNorm, NormFinder, and BestKeeper, were used to analyze the reference genes. ACTB expression was least affected by H₂O₂ treatment in TM cells, and the combination of PPIA and HPRT1 was the most suitable gene pair for normalization. GAPDH and TBP were the most unstable genes and accordingly should be avoided in experiments with TM cells. These results provide a foundation for analyses of the mechanisms underlying glaucoma, and emphasize the importance of selecting suitable reference genes for qPCR studies.     

Identification of suitable reference genes for expression studies in pomegranate under different biotic and abiotic stress conditions

Pomegranate (Punica granatum L.) is an important economic fruit crop, facing many biotic and abiotic challenges during cultivation. Several research programs are in progress to understand both biotic and abiotic stress factors and mitigate these challenges using gene expression studies based on the qPCR approach. However, research publications are not available yet to select the standard reference gene for normalizing target gene expression values in pomegranate. The most suitable candidate reference gene is required to ensure precise and reliable results for qPCR analysis. Eight candidate reference genes' stability was evaluated under different stress conditions using different algorithms such as ?Ct, geNorm, BestKeeper, NormFinder, and RefFinder. The various algorithms revealed that EFA1 and 18S rRNA were common and most stable reference genes (RGs) under abiotic and wilt stress. Whereas comprehensive ranking by RefFinder showed GAPDH and CYPF were the most stable RGs under combined biotic (pooled samples of all biotic stress) and bacterial blight samples. For normalizing target gene expression under wilt, nematode, bacterial blight, and abiotic stress conditions both GAPDH and CYPFreference genes are adequate for qPCR. The above data provide comprehensive details for the selection of a candidate reference gene in various stresses in pomegranate

     

Selection of reference genes for normalization of qRT-PCR analysis of differentially expressed genes in soybean exposed to cadmium

Accurate normalization of gene expression with qRT-PCR depends on the use of appropriate reference genes (RGs) for the species under a given set of experimental conditions. Multiple RGs for gene expression analysis of soybean exposed to heavy metal stress treatment have not been reported in the literature. In this study, we evaluated the expression stability of ten candidate RGs in leaves, roots and stems of two soybean cultivars exposed to cadmium (Cd). Based on the geNorm and NormFinder analysis, ACT3, PP2A, ELF1B and F-box were the most stable RGs in these gene expression studies. In contrast, G6PD, UBC2, TUB, and ELF1A were the most variable ones and should not be used as RGs in these experimental conditions.     

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。     

Carbohydrate accumulation may be the proximate trigger of anthocyanin biosynthesis under autumn conditions in Begonia semperflorens

Many plant leaves appear red in the autumn, and many papers have focused on the environmental factors and role of anthocyanin in this process. However few papers have examined the substances that are induced during this process. We hypothesised that excess sugar accumulation directly induces anthocyanin accumulation under autumn conditions. Using two methods (restricting phloem movement and exogenous sucrose feeding), we found that both surplus photosynthate and exogenous sucrose could induce anthocyanin biosynthesis, corresponding to up‐regulation of several enzymes involved in anthocyanin biosynthesis (phenylalanine ammonia lyase, chalcone isomerase, dihydroflavonol 4‐reductase and flavonoid 3‐O‐glucosyl transferase) and in transport (glutathione S‐transferase). Our results suggest that excess carbohydrate may be the proximate trigger for induction of anthocyanin biosynthesis in autumn, but only when carbohydrates are accumulated for storage.     

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples

Background  

Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals.     

梭梭qRT-PCR内参基因的筛选及稳定性分析

本研究旨在探明梭梭（Haloxylon ammodendron）在不同非生物胁迫下稳定表达的内参基因,为后续梭梭抗逆性相关基因功能研究奠定基础。研究采用实时定量聚合酶链式反应（qRT-PCR）技术从梭梭转录组数据库中检测了GAPDH、ef1-α、UBC、RPL32、ALB、50S-1721、50S-1063、RPⅡ、H3、PP2A、SOD、HSC70、TUA和TUB等14个候选内参基因在高温、干旱、盐、ABA和昼夜节律条件下的表达变化。利用geNorm、NormFinder、BestKeeper和RefFinder软件对梭梭候选内参基因的稳定性进行评价,最终筛选出合适的内参基因,并通过对梭梭磷酸烯醇丙酮酸羧化酶（phosphoenolpyruvate carboxylase, PEPC）基因表达分析,验证了不同内参基因对实验结果的影响。4种软件分析得到的最优内参基因存在差异,在RefFinder网站上综合排序分析表明,在ABA处理和昼夜节律下,ALB是最优内参基因,RPⅡ基因在干旱胁迫下表达最稳定,TUB和RPⅡ基因在盐胁迫下最适用,H3基因在高温胁迫下表达最为稳定。各种胁迫下最适宜的内参基因为ALB和RPL32。通过计算几何平均值,得到14个候选内参基因的综合稳定性排名,其中排名前两位的基因分别为SOD和RPL32。综上,RPL32和SOD可作为梭梭qRT-PCR标准化的内参基因。     

孟氏隐唇瓢虫qRT-PCR分析中内参基因的筛选

选择合适的内参基因是qRT-PCR研究的关键。本文以孟氏隐唇瓢虫Cryptolaemus montrouzieri Mulsant为研究材料,利用qRT-PCR技术,对孟氏隐唇瓢虫4个候选内参基因Actin、RPS23、GAPDH和β-tubulin的mRNA的表达量进行了分析,并用Ge Norm、Norm Finder和Best Keeper软件分析它们在孟氏隐唇瓢虫不同发育阶段及成虫不同组织中的表达稳定性。结果表明,以成虫不同组织为材料时,综合三种软件分析结果显示4个候选基因表达稳定性平均等级值排名为RPS23(rank=1)β-tubulin(rank=2.3)GAPDH(rank=3)Actin(rank=3.7),以不同发育时期虫体为材料时,综合分析结果显示4个候选内参基因表达稳定性平均等级值排名为RPS23(rank=1.7)Actin(rank=2)GAPDH(rank=2.7)β-tubulin(rank=3.7)。综合分析在瓢虫不同发育阶段及成虫不同组织两种处理下,三种软件的评价效果,4个候选基因表达稳定性等级值的总平均排名为RPS23(rank=1.3)Actin(rank=2.8)=GAPDH(rank=2.8)β-tubulin(rank=3)。RPS23在瓢虫不同发育阶段及成虫不同组织中均显示出较高的表达稳定性及与其它基因之间极大的相关性,可以确定为孟氏隐唇虫不同发育阶段及成虫不同组织基因表达分析中一个稳定表达的基因,可作为单个内参基因或者其它内参基因的协同基因,本实验为开展孟氏隐唇瓢虫功能基因表达分析奠定了方法学基础。