scholarly journals Methodological considerations on selection of stable reference genes for RT-qPCR in the neonatal rat brain in hypoxia and hypothermia

2019 ◽  
Author(s):  
M. Bustelo ◽  
M.A. Bruno ◽  
C.F. Loidl ◽  
H.W.M. Steinbusch ◽  
A.W.D. Gavilanes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rat Brain ◽  
Statistical Methods ◽  
Target Gene ◽  
Expression Profiles ◽  
Neonatal Rat ◽  
Experimental Conditions ◽  
Target Gene Expression ◽  
The Impact ◽  
Neonatal Rat Brain

AbstractReal-time reverse transcription PCR (qPCR) normalized to an internal reference gene (RG), is a frequently used method for quantifying gene expression changes in neuroscience. Although RG expression is assumed to be constantly independent of physiological or experimental conditions, several studies have shown that commonly used RGs are not expressed stably. The use of unstable RGs has a profound effect on the conclusions drawn from studies on gene expression, and almost universally results in spurious estimation of target gene expression. Approaches aimed at selecting and validating RGs often make use of different statistical methods, which may lead to conflicting results. The present study evaluates the expression of 5 candidate RGs (Actb, Pgk1, Sdha, Gapdh, Rnu6b) as a function of hypoxia exposure and hypothermic treatment in the neonatal rat cerebral cortex –in order to identify RGs that are stably expressed under these experimental conditions– and compares several statistical approaches that have been proposed to validate RGs. In doing so, we first analyzed the RG ranking stability proposed by several widely used statistical methods and related tools, i.e. the Coefficient of Variation (CV) analysis, GeNorm, NormFinder, BestKeeper, and the ΔCt method. Subsequently, we compared RG expression patterns between the various experimental groups. We found that these statistical methods, next to producing different rankings per se, all ranked RGs displaying significant differences in expression levels between groups as the most stable RG. As a consequence, when assessing the impact of RG selection on target gene expression quantification, substantial differences in target gene expression profiles were observed. As such, by assessing mRNA expression profiles within the neonatal rat brain cortex in hypoxia and hypothermia as a showcase, this study underlines the importance of further validating RGs for each new experimental paradigm considering the limitations of each selection method.

Effects of different endocrine disruptor (EDC) mixtures on gene expression in neonatal rat brain regions: Focus on developing excitatory synapses

2013 ◽  
Vol 221 ◽  
pp. S106
Author(s):  
Walter Lichtensteiger ◽  
Catherine Bassetti-Gaille ◽  
Oliver Faass ◽  
Julie Boberg ◽  
Sofie Christiansen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rat Brain ◽  
Endocrine Disruptor ◽  
Brain Regions ◽  
Neonatal Rat ◽  
Excitatory Synapses ◽  
Rat Brain Regions ◽  
Neonatal Rat Brain

NF?B activity and target gene expression in the rat brain after one and two exposures to ionizing radiation

1999 ◽  
Vol 7 (3) ◽  
pp. 145-152 ◽  
Author(s):  
Uma Raju ◽  
Glenice J. Gumin ◽  
Philip J. Tofilon
Keyword(s):  
Gene Expression ◽  
Ionizing Radiation ◽  
Rat Brain ◽  
Target Gene ◽  
Target Gene Expression

scholarly journals Primary endothelial cell–specific regulation of hypoxia‐inducible factor (HIF)‐1 and HIF‐2 and their target gene expression profiles during hypoxia

2019 ◽  
Vol 33 (7) ◽  
pp. 7929-7941 ◽  
Author(s):  
Rafal Bartoszewski ◽  
Adrianna Moszyńska ◽  
Marcin Serocki ◽  
Aleksandra Cabaj ◽  
Andreas Polten ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cell ◽  
Target Gene ◽  
Expression Profiles ◽  
Hypoxia Inducible Factor ◽  
Gene Expression Profiles ◽  
Target Gene Expression ◽  
Specific Regulation

scholarly journals Identification and Evaluation of Reference Genes for Normalization of Gene Expression in Developmental Stages, Sexes, and Tissues of Diaphania caesalis (Lepidoptera, Pyralidae)

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Zheng Wang ◽  
Qianqian Meng ◽  
Xi Zhu ◽  
Shiwei Sun ◽  
Aiqin Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Target Gene ◽  
Target Genes ◽  
Developmental Stages ◽  
Expression Profiles ◽  
Transcriptional Level ◽  
Internal Reference ◽  
Target Gene Expression ◽  
Qrt Pcr

Abstract Diaphania caesalis (Walker) is an important boring insect mainly distributed in subtropical and tropical areas and attacked tropical woody grain crops, such as starchy plants of Artocarpus. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful approach for investigating target genes expression profiles at the transcriptional level. However, the identification and selection of internal reference genes, which is often overlooked, is the most vital step before the analysis of target gene expression by qRT-PCR. So far, the reliable internal reference genes under a certain condition of D. caesalis have not been investigated. Therefore, this study evaluated the expression stability of eight candidate reference genes including ACT, β-TUB, GAPDH, G6PDH, RPS3a, RPL13a, EF1α, and EIF4A in different developmental stages, tissues and sexes using geNorm, NormFinder and BestKeeper algorithms. To verify the stability of the recommended internal reference genes, the expression levels of DcaeOBP5 were analyzed under different treatment conditions. The results indicated that ACT, RPL13a, β-TUB, RPS3a, and EF1α were identified as the most stable reference genes for further studies on target gene expression involving different developmental stages of D. caesalis. And ACT and EIF4A were recommended as stable reference genes for different tissues. Furthermore, ACT, EF1α, and RPS3a were ranked as the best reference genes in different sexes based on three algorithms. Our research represents the critical first step to normalize qRT-PCR data and ensure the accuracy of expression of target genes involved in phylogenetic and physiological mechanism at the transcriptional level in D. caesalia.

Effects of hypothydroidism on the gene expression of progesterone receptors in the neonatal rat brain

1994 ◽  
Vol 50 (5-6) ◽  
pp. 293-297 ◽  
Author(s):  
Shuji Hirata ◽  
Naoko Mouri-Yamada ◽  
Junzo Kato
Keyword(s):  
Gene Expression ◽  
Rat Brain ◽  
Progesterone Receptors ◽  
Neonatal Rat ◽  
Neonatal Rat Brain

AMPA-Induced suppression of oligodendroglial gene expression in neonatal rat brain

2001 ◽  
Vol 132 (2) ◽  
pp. 175-178 ◽  
Author(s):  
Haiyan Xu ◽  
John D.E. Barks ◽  
Yi-Qing Liu ◽  
Faye S. Silverstein
Keyword(s):  
Gene Expression ◽  
Rat Brain ◽  
Neonatal Rat ◽  
Neonatal Rat Brain

scholarly journals Hypoxic Preconditioning Induces Changes in HIF-1 Target Genes in Neonatal Rat Brain

2001 ◽  
Vol 21 (9) ◽  
pp. 1105-1114 ◽  
Author(s):  
Nicole M. Jones ◽  
Marcelle Bergeron
Keyword(s):  
Gene Expression ◽  
Rat Brain ◽  
Target Genes ◽  
Hypoxia Inducible Factor ◽  
Hypoxic Preconditioning ◽  
Neonatal Rat ◽  
Glucose Transporter 1 ◽  
Glut 1 ◽  
Hypoxia Inducible Factor 1 ◽  
Neonatal Rat Brain

Hypoxic preconditioning induces tolerance to hypoxic-ischemic injury in neonatal rat brain and is associated with changes in gene expression. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that is strongly induced by hypoxia or the hypoxia-mimetic compound cobalt chloride (CoCl2). Hypoxia-inducible factor-1 modulates the expression of several target genes including the glycolytic enzymes, glucose transporter-1 (GLUT-1), and erythropoietin. Recently, HIF-1 expression was shown to increase after hypoxic and CoCl2 preconditioning in newborn rat brain. To study the involvement of HIF-1 target genes in neonatal hypoxia-induced ischemic tolerance, the authors examined the brains of newborn rats after exposure to hypoxia (8% O2 for 3 hours) or injection of CoCl2 (60 mg/kg). Preconditioning with hypoxia or CoCl2 24 hours before hypoxia-ischemia afforded a 96% and 76% brain protection, respectively, compared with littermate control animals. Hypoxic preconditioning increased the expression of GLUT-1 mRNA and protein, and of aldolase, phosphofructokinase, and lactate dehydrogenase proteins but not mRNA. This suggests that the modulation of glucose transport and glycolysis by hypoxia may contribute to the development of hypoxia-induced tolerance. In contrast, preconditioning with CoCl2 did not produce any change in HIF-1 target gene expression suggesting that different molecular mechanisms may be involved in the induction of tolerance by hypoxia and CoCl2 in newborn brain.

Effect of fetal hypothyroidism on MyomiR network and its target gene expression profiles in heart of offspring rats

2017 ◽  
Vol 436 (1-2) ◽  
pp. 179-187 ◽  
Author(s):  
Nasibeh Yousefzadeh ◽  
Sajad Jeddi ◽  
Rafighe Ghiasi ◽  
Mohammad Reza Alipour
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Target Gene Expression ◽  
Fetal Hypothyroidism

scholarly journals Redesigned TetR-Aptamer System To Control Gene Expression in Plasmodium falciparum

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Krithika Rajaram ◽  
Hans B. Liu ◽  
Sean T. Prigge
Keyword(s):  
Gene Expression ◽  
Plasmodium Falciparum ◽  
Target Gene ◽  
Essential Genes ◽  
Untranslated Regions ◽  
Dependent Manner ◽  
Control Of Gene Expression ◽  
Content Type ◽  
Target Gene Expression ◽  
The Impact

ABSTRACT One of the most powerful approaches to understanding gene function involves turning genes on and off at will and measuring the impact at the cellular or organismal level. This particularly applies to the cohort of essential genes where traditional gene knockouts are inviable. In Plasmodium falciparum, conditional control of gene expression has been achieved by using multicomponent systems in which individual modules interact with each other to regulate DNA recombination, transcription, or posttranscriptional processes. The recently devised TetR-DOZI aptamer system relies on the ligand-regulatable interaction of a protein module with synthetic RNA aptamers to control the translation of a target gene. This technique has been successfully employed to study essential genes in P. falciparum and involves the insertion of several aptamer copies into the 3′ untranslated regions (UTRs), which provide control over mRNA fate. However, aptamer repeats are prone to recombination and one or more copies can be lost from the system, resulting in a loss of control over target gene expression. We rectified this issue by redesigning the aptamer array to minimize recombination while preserving the control elements. As proof of concept, we compared the original and modified arrays for their ability to knock down the levels of a putative essential apicoplast protein (PF3D7_0815700) and demonstrated that the modified array is highly stable and efficient. This redesign will enhance the utility of a tool that is quickly becoming a favored strategy for genetic studies in P. falciparum. IMPORTANCE Malaria elimination efforts have been repeatedly hindered by the evolution and spread of multidrug-resistant strains of Plasmodium falciparum. The absence of a commercially available vaccine emphasizes the need for a better understanding of Plasmodium biology in order to further translational research. This has been partly facilitated by targeted gene deletion strategies for the functional analysis of parasite genes. However, genes that are essential for parasite replication in erythrocytes are refractory to such methods, and require conditional knockdown or knockout approaches to dissect their function. One such approach is the TetR-DOZI system that employs multiple synthetic aptamers in the untranslated regions of target genes to control their expression in a tetracycline-dependent manner. Maintaining modified parasites with intact aptamer copies has been challenging since these repeats can be lost by recombination. By interspacing the aptamers with unique sequences, we created a stable genetic system that remains effective at controlling target gene expression.

scholarly journals Selection of Reference Genes for Normalization of Real-Time PCR Data in Calliptamus italicus (Orthoptera: Acrididae) Under Different Temperature Conditions

2019 ◽  
Vol 19 (6) ◽  
Author(s):  
Hongxia Hu ◽  
Xiaofang Ye ◽  
Han Wang ◽  
Rong Ji
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Expression Analysis ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Target Gene ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Target Gene Expression

Abstract Global warming has dominated worldwide climate change trends, and adaptability to high temperatures is the main factor underlying the spread of the pest Calliptamus italicus in Xinjiang Province, China. However, knowledge about the molecular mechanisms responsible for this adaptability and other related biological properties of C. italicus remain relatively unclear. Real-time quantitative polymerase chain reaction (RT-qPCR) is a key tool for gene expression analysis associated with various biological processes. Reference genes are necessary for normalizing gene expression levels across samples taken from specific experimental conditions. In this study, transcript level of five genes (GAPDH, 18S, TUB, ACT, and EF1α), commonly used as reference genes, were evaluated under nine different temperatures (27, 30, 33, 36, 39, 42, 45, 48, and 51°C) to assess their expression stability and further select the most suitable to be used on normalization of target gene expression data. Gene expression profiles were analyzed using geNorm, NormFinder, and BestKeeper software packages. The combined results demonstrated that the best-ranked reference genes for C. italicus are EF1α, GAPDH, and ACT under different thermal stress conditions. This is the first study that assesses gene expression analysis across a range of temperatures to select the most appropriate reference genes for RT-qPCR data normalization in C. italicus. These results should assist target gene expression analysis associated with heat stress in C. italicus.

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