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.     

Validation of reference genes for the relative quantification of gene expression in human epicardial adipose tissue

Background

Relative quantification is a commonly used method for assessing gene expression, however its accuracy and reliability is dependent upon the choice of an optimal endogenous control gene, and such choice cannot be made a priori. There is limited information available on suitable reference genes to be used for studies involving human epicardial adipose tissue. The objective of the current study was to evaluate and identify optimal reference genes for use in the relative quantification of gene expression in human epicardial fat depots of lean, overweight and obese subjects.

Methodology/Principal Findings

Some of the commonly used reference genes including 18S, ACTB, RPL27, HPRT, CYCA, GAPDH, RPLPO, POLR2A and B2M were quantified using real-time PCR analysis. The expression stability of these genes was evaluated using Genorm, Normfinder and Bestkeeper algorithms. In addition, the effect of sample size on the validation process was studied by randomly categorizing subjects in two cohorts of n = 2 and n = 33.

Conclusions/Significance

CYCA, GAPDH and RPL27 were identified as the most stable genes common to all three algorithms and both sample sizes. Their use as reference gene pairs might contribute to the enhanced robustness of relative quantification in the studies involving the human epicardial adipose tissue.     

Identification of reference genes for qRT-PCR in human lung squamous-cell carcinoma by RNA-Seq

Although the accuracy of quantitative real-time polymerase chain reaction （qRT-PCR） is highly dependent on the reliable reference genes, many commonly used reference genes are not stably expressed and as such are not suitable for quantification and normalization of qRT-PCR data. The aim of this study was to identify novel reliable reference genes in lung squamous-cell carcinoma. We used RNA sequencing （RNA-Seq） to survey the whole genome expression in 5 lung normal samples and 44 lung squamous-cell carcinoma samples. We evaluated the expression proffies of 15 commonly used reference genes and identified five additional candidate reference genes. To validate the RNA-Seq dataset, we used qRT-PCR to verify the expression levels of these 20 genes in a separate set of 100 pairs of normal lung tissue and lung squamous-cell carcinoma samples, and then analyzed these results using geNorm and NormFinder. With respect to 14 of the 15 common reference genes （B2M, GAPDH, GUSB, HMBS, HPRT1, IP08, PGK1, POLR2A, PPIA, RPLPO, TBP, TFRC, UBC, and YWHAZ）, the expression levels were either too low to be easily detected, or exhibited a high degree of variability either between lung normal and squamous-cell carcinoma samples, or even among samples of the same tissue type. In contrast, 1 of the 15 common reference genes （ACTB） and the 5 additional candidate reference genes （EEFIA1, FAU, RPS9, RPSll, and RPSI4） were stably and constitutively expressed at high levels in all the samples tested. ACTB, EEFIAI, FA U, RPS9, RPSl l, and RPS14 are ideal reference genes for qRT-PCR analysis of lung squamous-cell carcinoma, while 14 commonly used qRT-PCR reference genes are less appropriate in this context.     

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。     

Identification of qRT-PCR reference genes for analysis of opioid gene expression in a hibernator

Previous work has suggested that central and peripheral opioid signaling are involved in regulating torpor behavior and tissue protection associated with the hibernation phenotype. We used quantitative real-time PCR (qRT-PCR) to measure mRNA levels of opioid peptide precursors and receptors in the brain and heart of summer ground squirrels (Ictidomys tridecemlineatus) and winter hibernating squirrels in the torpid or interbout arousal states. The use of appropriate reference genes for normalization of qRT-PCR gene expression data can have profound effects on the analysis and interpretation of results. This may be particularly important when experimental subjects, such as hibernating animals, undergo significant morphological and/or functional changes during the study. Therefore, an additional goal of this study was to identify stable reference genes for use in qRT-PCR studies of the 13-lined ground squirrel. Expression levels of 10 potential reference genes were measured in the small intestine, liver, brain, and heart, and the optimal combinations of the most stable reference genes were identified by the GeNorm Excel applet. Based on this analysis, we provide recommendations for reference genes to use in each tissue that would be suitable for comparative studies among different activity states. When appropriate normalization of mRNA levels was used, there were no changes in opioid-related genes in heart among the three activity states; in brain, DOR expression was highest during torpor, lowest in interbout arousal and intermediate in summer. The results support the idea that changes in DOR expression may regulate the level of neuronal activity in brain during the annual hibernation cycle and may contribute to hibernation-associated tissue protection.     

黑木耳实时荧光定量PCR内参基因的筛选

黑木耳Auricularia heimuer是我国主要栽培的食用菌之一,具有较高的经济价值和生态价值。本研究以黑木耳不同菌株（A14、A137和A12）和不同生育期的样品（菌丝体、原基和子实体）为实验材料,提取RNA,反转录成cDNA,在全基因组注释结果的基础上,选择12个候选内参基因（APRTase、β-TUB、RPL2、EF-1a、EF-2、PGI、PGM、H ⁺-ATPase、Tspd、TUB-1a、18S rRNA和28S rRNA）并设计跨内含子的引物,采用实时荧光定量PCR（qRT-PCR）技术进行扩增,利用geNorm、NormFinder、BestKeeper和ΔCt算法以及综合评价软件RefFinder,筛选适宜的内参基因。结果表明18S rRNA、β-TUB、EF1-a和28S rRNA适宜作为不同菌株的内参基因,APRTase、18S rRNA和28S rRNA适宜作为不同生育期的内参基因。     

Selection of suitable reference genes for qRT-PCR analysis of Begonia semperflorens under stress conditions

Molecular Biology Reports - Begonia semperflorens (B. semperflorens), belonging to the family Begoniaceae, has now been widely cultivated worldwide and is famous for its ornamental plants with...     

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...     

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.     

Expression of clock genes in human subcutaneous and visceral adipose tissues

Disrupted circadian rhythms are associated with obesity and metabolic alterations, but little is known about the participation of peripheral circadian clock machinery in these processes. The aim of the present study was to analyze RNA expression of clock genes in subcutaneous (SAT) and visceral (VAT) adipose tissues of male and female subjects in AM (morning) and PM (afternoon) periods, and its interactions with body mass index (BMI). Ninety-one subjects (41 ± 11 yrs of age) presenting a wide range of BMI (21.4 to 48.6 kg/m(2)) were included. SAT and VAT biopsies were obtained from patients undergoing abdominal surgeries. Clock genes expressions were evaluated by qRT-PCR. The only clock gene that showed higher expression (p 相似文献   

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。     

Evaluation of potential reference genes for qRT-PCR data normalization in HeLa cells

Reference genes selection is one of the most important stages in qPCR data normalization when a problem of quantitative determination of gene expression is addressed. Stability of gene expression level in all experimental conditions is a basic criterion for the reference gene selection. Over the past decade a lot of publications concerning validation methods of suitable reference genes appeared. In this paper, the main approaches (ΔCt, geNorm, qBase and Haller’s equivalence test) were applied for the reference genes identification in HeLa cell line which is one of the most popular cellular models. Expression stability of seven candidate genes (HPRT1, ACTB, GAPDH, RPS18, HSPC3, UBC and SDHA) was determined at standard conditions, under heat shock and during relaxation. The genes RPS18 and HSPC3 were chosen as reference after the combination of all the validation methods.     

Validation of endogenous control genes in human adipose tissue: relevance to obesity and obesity-associated type 2 diabetes mellitus.

The aim of the present study was to test the influence of obesity and the presence of type 2 diabetes mellitus (T2DM) on the expression of ten housekeeping genes and of the 18S rRNA in a group of human adipose tissue samples from the omental and subcutaneous depot. Adipose tissue biopsies were obtained by laparoscopic surgery from lean and obese patients. After the extraction, mRNA levels in adipose tissue samples were quantified by real-time PCR using the commercial HUMAN ENDOGENOUS CONTROL PLATES. From the genes analyzed, 18S rRNA exhibited the most stable expression levels in both depots regardless of the pathophysiological conditions of obesity and obesity-associated T2DM. Contrarily, GAPD was the gene with the highest variation in its expression levels, being upregulated (8.0-fold) in the obese group and downregulated (3.5-fold) in obesity-associated T2DM. Our results show that 18S rRNA may be the most suitable gene for normalization in expression studies performed in human adipose tissue samples obtained from patients suffering from obesity and/or obesity-associated T2DM, whereas GAPD is less appropriate for comparison purposes under these circumstances.