共查询到20条相似文献,搜索用时 31 毫秒
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《Journal of plant physiology》2014,171(16):1541-1544
Quantitative PCR studies need proper reference genes with expression stability exclusively validated under certain experimental conditions. The expression stability of several genes commonly used as references was tested under 24-epibrassinolide (EBR) and temperature treatment. Different statistical approaches (qBasePLUS, BestKeeper, NormFinder) were used to prepare rankings of expression stability in two species of an economic importance: common wheat (Triticum aestivum) and perennial ryegrass (Lolium perenne). Candidate reference genes were shown to be regulated differentially in these two plant species. The maximum stability values indicated that the expression stability was higher in T. aestivum. Taking into account of all ranks it seems that TBP-1 and UBI in ryegrass and ACT, ADP and EF1A in wheat should be used as reference genes in the brassinosteroids and temperature involving studies. 相似文献
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Background
Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development.Methodology/Principal Findings
We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed.Conclusion
Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot. 相似文献3.
Tianyi Li Hongying Diao Lei Zhao Yue Xing Jichang Zhang Ning Liu Youyou Yan Xin Tian Wei Sun Bin Liu 《BMC molecular biology》2017,18(1):10
Background
Oxidative stress can induce cell injury in vascular endothelial cells, which is the initial event in the development of atherosclerosis. Although quantitative real-time polymerase chain reaction (qRT-PCR) has been widely used in gene expression studies in oxidative stress injuries, using carefully validated reference genes has not received sufficient attention in related studies. The objective of this study, therefore, was to select a set of stably expressed reference genes for use in qRT-PCR normalization in oxidative stress injuries in human umbilical vein endothelial cells (HUVECs) induced by hydrogen peroxide (H2O2).Results
Using geNorm analysis, we found that five stably expressed reference genes were sufficient for normalization in qRT-PCR analysis in HUVECs treated with H2O2. Genes with the most stable expression according to geNorm were U6, TFRC, RPLP0, GAPDH, and ACTB, and according to NormFinder were ALAS1, TFRC, U6, GAPDH, and ACTB.Conclusion
Taken together, our study demonstrated that the expression stability of reference genes may differ according to the statistical program used. U6, TFRC, RPLP0, GAPDH, and ACTB was the optimal set of reference genes for studies on gene expression performed by qRT-PCR assays in HUVECs under oxidative stress study.4.
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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. 相似文献8.
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Jaime Ramos-Cejudo María Gutiérrez-Fernández Berta Rodríguez-Frutos Mercedes Expósito Alcaide Fátima Sánchez-Cabo Ana Dopazo Exuperio Díez–Tejedor 《PloS one》2012,7(12)
Background
A large number of genes are regulated to promote brain repair following stroke. The thorough analysis of this process can help identify new markers and develop therapeutic strategies. This study analyzes gene expression following experimental stroke.Methodology/Principal Findings
A microarray study of gene expression in the core, periinfarct and contralateral cortex was performed in adult Sprague-Dawley rats (n = 60) after 24 hours (acute phase) or 3 days (delayed stage) of permanent middle cerebral artery (MCA) occlusion. Independent qRT-PCR validation (n = 12) was performed for 22 of the genes. Functional data were evaluated by Ingenuity Pathway Analysis. The number of genes differentially expressed was 2,612 (24 h) and 5,717 (3 d) in the core; and 3,505 (24 h) and 1,686 (3 d) in the periinfarct area (logFC>|1|; adjP<0.05). Expression of many neurovascular unit development genes was altered at 24 h and 3 d including HES2, OLIG2, LINGO1 and NOGO-A; chemokines like CXCL1 and CXCL12, stress-response genes like HIF-1A, and trophic factors like BDNF or BMP4. Nearly half of the detected genes (43%) had not been associated with stroke previously.Conclusions
This comprehensive study of gene regulation in the core and periinfarct areas at different times following permanent MCA occlusion provides new data that can be helpful in translational research. 相似文献15.
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