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.     

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.     

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.     

Selection of suitable reference genes for real-time PCR studies of Atlantic halibut development

Gene expression studies are fundamental to understand the molecular basis of severe malformations in fish development, particularly under aquaculture conditions. Real-time PCR (qPCR) is the most accurate method of quantifying gene expression, provided that suitable endogenous controls are used to normalize the data. To date, no reference genes have been validated for developmental gene expression studies in Atlantic halibut (Hippoglossus hippoglossus). We have determined the expression profiles of 6 candidate reference genes (Actb, Eef2, Fau, Gapdh, Tubb2 and 18S rRNA) in 6 embryonic and 5 larval stages of Atlantic halibut development. There were significant changes in expression levels throughout development, which stress the importance and complexity of finding appropriate reference genes. The three software applications (BestKeeper, geNorm and NormFinder) used to evaluate the stability of potential reference genes produced comparable results. Tubb2 and Actb were the most stable genes across the different developmental stages, whereas 18S rRNA and Gapdh were the most variable genes and thus inappropriate to use as reference genes. According to geNorm and NormFinder, the best two-gene normalization factors corresponded to the geometric average of Tubb2/Actb and Tbb2/Fau, respectively. We believe that either of these normalization factors can be used for future developmental gene expression studies in Atlantic halibut.     

Identification of suitable reference genes for expression profiling studies using qRT-PCR in an important insect pest,Maruca vitrata

Molecular Biology Reports - Maruca vitrata is one of the potential insect pests that cause devastating losses to legume cultivation worldwide. Gene functional studies facilitate dissecting the...