scholarly journals The Importance of the Selection of Appropriate Reference Genes for Gene Expression Profiling in Adrenal Medulla or Sympathetic Ganglia of Spontaneously Hypertensive Rat

2016 ◽  
pp. 401-411 ◽  
Author(s):  
A. VAVŘÍNOVÁ ◽  
M. BEHULIAK ◽  
J. ZICHA
Keyword(s):  
Mrna Expression ◽  
Adrenal Medulla ◽  
Reference Gene ◽  
Internal Control ◽  
Reference Genes ◽  
Sympathetic Ganglia ◽  
Stable Reference Gene ◽  
Spontaneously Hypertensive ◽  
Wky Rats ◽  
Catecholaminergic System

Catecholaminergic system plays an important role in hypertension development. The available results on mRNA expression of catecholaminergic system genes in spontaneously hypertensive rats (SHR) are often contradictory. One of the possible causes might be the use of various reference genes as internal controls. In the present study, we searched for suitable reference genes in adrenal medulla or sympathetic ganglia of SHR and Wistar-Kyoto (WKY) rats, which would enable reliable comparison of mRNA expression between these two strains. The mRNA expression was measured by quantitative real-time PCR in adrenal medulla and superior cervical ganglia of 4-week-old or 24-week-old SHR and WKY rats. We evaluated 12 reference genes by three software tools (Normfinder, BestKeeper, geNorm) and compared them for the standardization of mRNA expression. Combination of reference genes Hprt1 and Ywhaz in adrenal medulla and Gapdh and 18S in sympathetic ganglia were chosen as the best ones. 18S was found as applicable reference gene in both tissues. We found many alterations in expression of catecholaminergic system genes in adrenal medulla and sympathetic ganglia of SHR. The usage of the most or the least stable reference gene as internal control changed results moderately in sympathetic ganglia but seriously in adrenal medulla. For example, tyrosine hydroxylase (Th) gene was underexpressed in adrenal medulla of adult SHR using the appropriate reference gene but unchanged after the standardization to the least stable reference gene. Our results indicate the importance of appropriate internal control. The suitability of reference genes should be checked again in the case of change in experimental conditions.

scholarly journals Reference Gene Selection for qPCR Normalization ofKosteletzkya virginicaunder Salt Stress

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Xiaoli Tang ◽  
Hongyan Wang ◽  
Chuyang Shao ◽  
Hongbo Shao
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Selection ◽  
Expression Profiles ◽  
Elongation Factor ◽  
Gene Expression Profiles ◽  
Stable Reference Gene ◽  
Experimental Conditions ◽  
Suitable Reference

Kosteletzkya virginica(L.) is a newly introduced perennial halophytic plant. Presently, reverse transcription quantitative real-time PCR (qPCR) is regarded as the best choice for analyzing gene expression and its accuracy mainly depends on the reference genes which are used for gene expression normalization. In this study, we employed qPCR to select the most stable reference gene inK. virginicawhich showed stable expression profiles under our experimental conditions. The candidate reference genes were 18S ribosomal RNA (18SrRNA),β-actin (ACT),α-tubulin (TUA), and elongation factor (EF). We tracked the gene expression profiles of the candidate genes and analyzed their stabilities through BestKeeper, geNorm, and NormFinder software programs. The results of the three programs were identical and18SrRNAwas assessed to be the most stable reference gene in this study. However,TUAwas identified to be the most unstable. Our study proved again that the traditional reference genes indeed displayed a certain degree of variations under given experimental conditions. Importantly, our research also provides guidance for selecting most suitable reference genes and lays the foundation for further studies inK. virginica.

scholarly journals Toxicity Evaluation and Biomarker Selection with Validated Reference Gene in Embryonic Zebrafish Exposed to Mitoxantrone

2018 ◽  
Vol 19 (11) ◽  
pp. 3516 ◽  
Author(s):  
Lili Liu ◽  
Hua Zhu ◽  
Yanchun Yan ◽  
Peng Lv ◽  
Wei Wu
Keyword(s):  
Reference Gene ◽  
Internal Control ◽  
Reference Genes ◽  
Zebrafish Embryo ◽  
Zebrafish Embryos ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Spontaneous Movement ◽  
Clinical Value ◽  
Potential Biomarker

Notwithstanding the widespread use and promising clinical value of chemotherapy, the pharmacokinetics, toxicology, and mechanism of mitoxantrone remains unclear. To promote the clinical value in the treatment of human diseases and the exploration of potential subtle effects of mitoxantrone, zebrafish embryos were employed to evaluate toxicity with validated reference genes based on independent stability evaluation programs. The most stable and recommended reference gene was gapdh, followed by tubα1b, for the 48 h post fertilization (hpf) zebrafish embryo mitoxantrone test, while both eef1a1l1 and rpl13α were recommended as reference genes for the 96 hpf zebrafish embryo mitoxantrone test. With gapdh as an internal control, we analyzed the mRNA levels of representative hepatotoxicity biomarkers, including fabp10a, gclc, gsr, nqo1, cardiotoxicity biomarker erg, and neurotoxicity biomarker gfap in the 48 hpf embryo mitoxantrone test. The mRNA levels of gclc, gsr, and gfap increased significantly in 10 and 50 μg/L mitoxantrone-treated 48 hpf embryos, while the transcript levels of fabp10a decreased in a dose-dependent manner, indicating that mitoxantrone induced hepatotoxicity and neurotoxicity. Liver hematoxylin–eosin staining and the spontaneous movement of embryos confirmed the results. Thus, the present research suggests that mitoxantrone induces toxicity during the development of the liver and nervous system in zebrafish embryos and that fabp10a is recommended as a potential biomarker for hepatotoxicity in zebrafish embryos. Additionally, gapdh is proposed as a reference gene for the 48 hpf zebrafish embryo mitoxantrone toxicity test, while eef1a1l1 and rpl13α are proposed as that for the 96 hpf test.

scholarly journals Identification and validation of potential reference gene for effective dsRNA knockdown analysis in Chilo partellus

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Olawale Samuel Adeyinka ◽  
Bushra Tabassum ◽  
Idrees Ahmad Nasir ◽  
Iqra Yousaf ◽  
Imtiaz Ahmad Sajid ◽  
...  
Keyword(s):  
Reference Gene ◽  
Chilo Partellus ◽  
Reference Genes ◽  
Gene Selection ◽  
Developmental Stages ◽  
Stable Reference Gene ◽  
Suitable Reference Gene ◽  
Potential Reference Gene ◽  
Alternate Control ◽  
Suitable Reference

Abstract Chilo partellus is an invasive polyphagous pest that has not been effectively managed with chemical pesticides. To select potential dsRNAs for use in an alternate control strategy, it is crucial to identify and evaluate stable reference genes for knockdown expression studies. This study evaluates the expression stability of seven candidate reference genes in C. partellus larvae fed on crude bacterially-expressed dsRNAs and purified dsRNAs at different time intervals, as well as the developmental stages and sexes. The expression stabilities of the reference genes were evaluated with different software programmes, such as BestKeeper, NormFinder, deltaCt, geNorm, and RefFinder. The overall results rank ELF as the most stably expressed reference gene when larvae were fed with crude bacteria-induced dsRNAs and purified dsRNA. However, Tubulin and HSP70 were more stable under different developmental stages and sexes. The expression levels of larvae that were fed crude bacteria-induced dsRNAs of Chitinase and Acetylcholinesterase were normalized with the four most stable reference genes (ELF, HSP70, V-ATPase and Tubulin) and the least stable reference gene (18S and HSP70) based on the geNorm algorithm. The least stable reference gene showed inconsistent knockdown expression, thereby confirming that the validation of a suitable reference gene is crucial to improve assay accuracy for dsRNA-targeted gene selection in C. partellus.

scholarly journals iRGvalid: A Robust in silico Method for Optimal Reference Gene Validation

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhongxu Zhu ◽  
Keqin Gregg ◽  
Wenli Zhou
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
In Silico ◽  
Reference Genes ◽  
Target Gene ◽  
Stable Reference Gene ◽  
Expression Data ◽  
Reference Gene Validation ◽  
Cancer Types ◽  
The Stability

BackgroundAppropriate reference genes are critical to accurately quantifying relative gene expression in research and clinical applications. Numerous efforts have been made to select the most stable reference gene(s), but a consensus has yet to be achieved. In this report, we propose an in silico reference gene validation method, iRGvalid, that can be used as a universal tool to validate the reference genes recommended from different resources so as to identify the best ones without a need for any wet lab validation tests.MethodsiRGvalid takes advantage of high throughput gene expression data and is built on a double-normalization strategy. First, the expression level of each individual gene is normalized against the total gene expression level of each sample, followed by a target gene normalization to the candidate reference gene(s). Linear regression analysis is then performed between the pre- and post- normalized target gene across the whole sample set to evaluate the stability of the reference gene(s), which is positively associated with the Pearson correlation coefficient, Rt. The higher the Rt value, the more stable the reference gene. We applied iRGvalid to 14 candidate reference genes to validate and identify the most stable reference genes in four cancer types: lung adenocarcinoma, breast cancer, colon adenocarcinoma, and nasopharyngeal cancer. The stability of the reference gene is evaluated both individually and in groups of all possible combinations.ResultsHighly stable reference genes resulted in high Rt values regardless of the target gene used. The highest stability was achieved with a specific combination of 3 to 6 reference genes. A few genes were among the best reference genes across the cancer types studied here.ConclusioniRGvalid provides an easy and robust method to validate and identify the most stable reference gene or genes from a pool of candidate reference genes. The inclusivity of large expression data sets as well as the direct comparison of candidate reference genes makes it possible to identify reference genes with universal quality. This method can be used in any other gene expression studies when large cohorts of expression data are available.

Identification of Suitable Internal Control Genes for Quantitative Real-Time PCR Expression Analyses in Peanut (Arachis hypogaea)

2010 ◽  
Vol 37 (1) ◽  
pp. 12-19 ◽  
Author(s):  
Yael Brand ◽  
Ran Hovav
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Developmental Stages ◽  
Peanut Kernel ◽  
Tissue Samples ◽  
The Stability

Abstract Real-time qPCR is currently the most sensitive technique available for the detection of low-level mRNA expression. For more reliable and precise gene expression analyses, real-time PCR data for a sequence of interest must be normalized against that of a control gene, which is uniformly expressed in various tissues and during different phases of development. So far, suitable internal controls for gene expression studies in peanut have not been identified. We assessed the expression of 10 frequently used housekeeping genes, specifically ubq10, gapdh, hel1, yls8, 14-3-3, 60s, ubc, ef-1α, act7, and adh3. Using the algorithms available through the GeNorm and NormFinder programs, the stability of their expression was estimated in a set of five diverse peanut tissue samples derived from a Virginia-type peanut cultivar (Shulamit). Collectively, the gene with the most stable expression across all of the examined tissues and both programs was adh3, followed by 60s and yls8, which had minimal estimated intra- and inter-tissue variation. The stability of two stable reference genes (adh3 and yls8) compared with two less stable (14-3-3 and ubq10) reference genes was validated in unpooled tissue samples from five peanut kernel developmental stages. Finally, the effect of the use of one or more reference genes on the observed relative expression levels of an important seed oil metabolism gene, diacylglycerol acyltransferase 1 (Dgat1), during kernel development was demonstrated. Based on findings, the suggestion is that adh3, or a combination of this gene with 60s and yls8 should be considered for use in quantitative mRNA expression analyses in Arachis, particularly in studies involving seed development; whereas ubq10 and gapdh should be avoided.

scholarly journals Reference Gene Validation Within the Rat Brain Under Mild Intermittent Ketosis Induced by Supplementation with Medium-Chain Triglycerides

2021 ◽  
Author(s):  
Alexander Pavlovich Schwarz ◽  
Veronika Alexandrovna Nikitina ◽  
Darya Uladzimirawna Krytskaya ◽  
Ksenia Pavlovna Shcherbakova ◽  
Alexander Nikolaevich Trofimov
Keyword(s):  
Prefrontal Cortex ◽  
Mrna Expression ◽  
Medial Prefrontal Cortex ◽  
Reference Gene ◽  
Reference Genes ◽  
Dorsal Hippocampus ◽  
Housekeeping Genes ◽  
Ventral Hippocampus ◽  
Medium Chain Triglycerides ◽  
Medium Chain

Abstract RT-qPCR has become the gold standard in mRNA expression analysis. However, it requires an accurate choice of reference genes for adequate normalization. This work aims to validate the reference genes for qPCR experiments in the brain of rats in the model of mild ketosis established through supplementation with medium-chain triglycerides (MCT) and intermittent fasting. The standard chow of adult Wistar rats was supplemented with MCT oil (2 ml/kg, i.g., during 6 h of fasting) or water (equivolume) for 1 month. The mRNA expression of 9 housekeeping genes (Actb, B2m, Gapdh, Hprt1, Pgk1, Ppia, Rpl13a, Sdha, Ywhaz) in the medial prefrontal cortex, dorsal and ventral hippocampus was measured by RT-qPCR. Using the RefFinder® online tool, we have found that the reference gene stability ranking strongly depended on the analyzed brain region. The most stably expressed reference genes were found to be Ppia, Actb, and Rpl13a in the medial prefrontal cortex; Rpl13a, Ywhaz, and Pgk1 in the dorsal hippocampus; Ywhaz, Sdha, and Ppia in the ventral hippocampus. The B2m was identified as an invalid reference gene in the ventral hippocampus, while Sdha, Actb, and Gapdh were unstable in the dorsal hippocampus. The stabilities of the examined housekeeping genes were lower in the dorsal hippocampus compared to the ventral hippocampus and the medial prefrontal cortex. Thus, the expression stability of reference genes strongly depends on the examined brain regions. The dorsal and ventral hippocampal areas differ in reference genes stability rankings, which should be taken into account in the RT-qPCR experimental design.

Differences In Utero in Activities of Catecholamine Biosynthetic Enzymes in Adrenals of Spontaneously Hypertensive Rats

1981 ◽  
Vol 61 (s7) ◽  
pp. 227s-230s ◽  
Author(s):  
G. Teitelman ◽  
R. A. Ross ◽  
T. H. Joh ◽  
D. J. Reis
Keyword(s):  
Adrenal Medulla ◽  
Spontaneously Hypertensive Rats ◽  
Sympathetic Ganglia ◽  
Biosynthetic Enzymes ◽  
Sprague Dawley ◽  
Hypertensive Rats ◽  
Spontaneously Hypertensive ◽  
Catecholamine Biosynthesis ◽  
Sd Rats ◽  
Wistar Kyoto

1. We sought to determine if catecholamine biosynthetic enzymes of spontaneously hypertensive rats (SHR) differed from those of normotensive Wistar—Kyoto (WKY) and Sprague—Dawley (SD) control rats before birth. 2. By immunocytochemical and biochemical methods we compared strains for the time of appearance and maturation of the enzymes tyrosine hydroylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) in sympathetic ganglia and adrenals. 3. The time of appearance of enzymes was identical in all three strains: TH and DBH first appeared in sympathetic ganglia on embryonic day 11 (E11) and in adrenal medulla on E16. PNMT, restricted to adrenal medulla, appeared later on E18. 4. The activity of adrenal TH prenatally on E18 and E21 and at day of birth (P1) in SHR was approximately two fold that in WKY or SD rats. In contrast PNMT was lower in SHR but only on E18. 5. Thus, although the timing of the first expression of adrenergic phenotypes is similar in SHR and normotensive controls, the differences in TH activity in adrenals suggest an enhanced biosynthetic capacity for catecholamines in this strain before birth. 6. We conclude that SHR differ from normotensive rats from the first expression of some of the genes controlling catecholamine biosynthesis.

Kinin B1 and B2 receptor mRNA expression in the hypothalamus of spontaneously hypertensive rats

2002 ◽  
Vol 80 (4) ◽  
pp. 258-263 ◽  
Author(s):  
F Qadri ◽  
W Häuser ◽  
O Jöhren ◽  
P Dominiak
Keyword(s):  
Mrna Expression ◽  
Spontaneously Hypertensive Rats ◽  
Mrna Levels ◽  
Hypertensive Rats ◽  
Kinin System ◽  
Spontaneously Hypertensive ◽  
Receptor Mrna ◽  
Wky Rats ◽  
Wistar Kyoto ◽  
Kallikrein Kinin System

The central hypertensive effects induced by bradykinin are known to be mediated via B2 receptors, which are present constitutively in the brain. B1 receptors are rapidly upregulated during inflammation, hyperalgesia, and experimental diabetes. The hypothalamus plays an important role in the regulation of cardiovascular homeostasis, and all components of kallikrein–kinin system have been identified in this area. Therefore, we analyzed the mRNA expression of B1 and B2 receptors in the hypothalamus of spontaneously hypertensive rats (SHR) by RT-PCR. Male SHR were studied at three different ages corresponding to the three phases in the development of hypertension: (i) 3–4 (prehypertensive), (ii) 7–8 (onset of hypertension), and (iii) 12–13 weeks (established hypertension) after birth, and compared with age-matched Wistar–Kyoto (WKY) rats. At all ages tested, B2 receptor mRNA levels in the hypothalamus of SHR were higher than age-matched WKY rats (p < 0.001). However, the B1 receptor mRNA levels were higher at the established phase of hypertension only. We conclude that B1 and B2 receptor mRNA are differentially expressed in the hypothalamus of SHR and may play different roles in the pathogenesis of hypertension: upregulation of B2 receptor mRNA from early age may participate in the pathogenesis of hypertension, whereas an upregulation of B1 receptor mRNA in the established phase of hypertension may reflect an epiphenomenon in essential hypertension.Key words: kinin receptors, mRNA expression, hypothalamus, SHR, WKY.

Sign in / Sign up

Export Citation Format

Share Document