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Taniya Mitra, Ivana Bilic, Michael Hess, Dieter Liebhart
Evaluation of reference genes for expression studies in chickens and turkeys is very much limited and unavailable for various infectious models. In this study, eight candidate reference genes HMBS, HPRT1, TBP, VIM, TFRC, RPLP0, RPL13 and RPS7 were evaluated by five different algorithms (GeNorm, NormFinder, BestKeeper©, delta CT, RefFinder) to assess their stability. In order to analyze a broad variation of tissues, spleen, liver, caecum and caecal tonsil of different aged specific pathogen free (SPF) layer chickens and commercial turkeys, uninfected or infected with the extracellular pathogen Histomonas meleagridis, were included...
October 20, 2016: Veterinary Research
Jianbo Li, Huixia Jia, Xiaojiao Han, Jin Zhang, Pei Sun, Mengzhu Lu, Jianjun Hu
Salix psammophila is a desert shrub willow that has extraordinary adaptation to abiotic stresses and plays an important role in maintaining local ecosystems. Moreover, S. psammophila is regarded as a promising biomass feedstock because of its high biomass yields and short rotation coppice cycle. However, few suitable reference genes (RGs) for quantitative real-time polymerase chain reaction (qRT-PCR) constrain the study on normalization of gene expression in S. psammophila until now. Here, we investigated the expression stabilities of 14 candidate RGs across tissue types and under four abiotic stress treatments, including heat, cold, salt, and drought treatments...
2016: Frontiers in Plant Science
Yanqin Niu, Yike Wu, Jinyong Huang, Qing Li, Kang Kang, Junle Qu, Furong Li, Deming Gou
Quantitative real-time PCR (qPCR) is the most frequently used method for measuring expression levels of microRNAs (miRNAs), which is based on normalization to endogenous references. Although circulating miRNAs have been regarded as potential non-invasive biomarker of disease, no study has been performed so far on reference miRNAs for normalization in colorectal cancer. In this study we tried to identify optimal reference miRNAs for qPCR analysis across colorectal cancer patients and healthy individuals. 485 blood-derived miRNAs were profiled in serum sample pools of both colorectal cancer and healthy control...
October 19, 2016: Scientific Reports
Yasin Panahi, Fahimeh Salasar Moghaddam, Zahra Ghasemi, Mandana Hadi Jafari, Reza Shervin Badv, Mohamad Reza Eskandari, Mehrdad Pedram
Childhood autism is a severe form of complex genetically heterogeneous and behaviorally defined set of neurodevelopmental diseases, collectively termed as autism spectrum disorders (ASD). Reverse transcriptase quantitative real-time PCR (RT-qPCR) is a highly sensitive technique for transcriptome analysis, and it has been frequently used in ASD gene expression studies. However, normalization to stably expressed reference gene(s) is necessary to validate any alteration reported at the mRNA level for target genes...
October 12, 2016: International Journal of Molecular Sciences
Anna Vavřínová, Michal Behuliak, Josef Zicha
OBJECTIVE: Catecholaminergic system plays an important role in blood pressure regulation and hypertension development. The available information concerning mRNA expression of catecholaminergic system genes in spontaneously hypertensive rats (SHR) are often contradictory. This might be due to various reference genes used as internal controls. We therefore searched for suitable reference genes for gene expression profiling in adrenal medulla and sympathetic ganglia of SHR and Wistar-Kyoto (WKY) rats, which would enable reliable comparison of mRNA expression of genes of catecholaminergic system between these strains...
September 2016: Journal of Hypertension
Anoeska Agatha Alida van de Moosdijk, Renée van Amerongen
Cell growth and differentiation are often driven by subtle changes in gene expression. Many challenges still exist in detecting these changes, particularly in the context of a complex, developing tissue. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) allows relatively high-throughput evaluation of multiple genes and developmental time points. Proper quantification of gene expression levels by qRT-PCR requires normalization to one or more reference genes. Traditionally, these genes have been selected based on their presumed "housekeeping" function, with the implicit assumption that they are stably expressed over the entire experimental set...
October 18, 2016: Scientific Reports
Weifang Wu, Qin Deng, Pibiao Shi, Jinghua Yang, Zhongyuan Hu, Mingfang Zhang
Watermelon (Citrullus lanatus) is a globally important crop belonging to the family Cucurbitaceae. The grafting technique is commonly used to improve its tolerance to stress, as well as to enhance its nutrient uptake and utilization. It is believed that miRNA is most likely involved in its nutrient-starvation response as a graft-transportable signal. The quantitative real-time reverse transcriptase polymerase chain reaction is the preferred method for miRNA functional analysis, in which reliable reference genes for normalization are crucial to ensure the accuracy...
2016: PloS One
Meng-Yao Li, Xiong Song, Feng Wang, Ai-Sheng Xiong
Parsley, one of the most important vegetables in the Apiaceae family, is widely used in the food, medicinal, and cosmetic industries. Recent studies on parsley mainly focus on its chemical composition, and further research involving the analysis of the plant's gene functions and expressions is required. qPCR is a powerful method for detecting very low quantities of target transcript levels and is widely used to study gene expression. To ensure the accuracy of results, a suitable reference gene is necessary for expression normalization...
2016: Frontiers in Plant Science
Carmen Rueda-Martínez, M Carmen Fernández, María Teresa Soto-Navarrete, Manuel Jiménez-Navarro, Ana Carmen Durán, Borja Fernández
Bicuspid aortic valve (BAV) is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40%) incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans...
2016: PloS One
Yasmina E Hernandez-Santana, Eduardo Ontoria, Ana C Gonzalez-García, M Antonieta Quispe-Ricalde, Vicente Larraga, Basilio Valladares, Emma Carmelo
The interaction of Leishmania with BALB/c mice induces dramatic changes in transcriptome patterns in the parasite, but also in the target organs (spleen, liver…) due to its response against infection. Real-time quantitative PCR (qPCR) is an interesting approach to analyze these changes and understand the immunological pathways that lead to protection or progression of disease. However, qPCR results need to be normalized against one or more reference genes (RG) to correct for non-specific experimental variation...
2016: PloS One
Zhongyang Sun, Jia Deng, Haizhen Wu, Qiyao Wang, Yuanxing Zhang
Edwardsiella tarda is a Gram-negative pathogenic bacterium in aquaculture which can cause hemorrhagic septicemia in fish. Many secreted proteins have been identified as virulent factors of E. tarda. While the virulent phenotype is based on the expression regulation of the virulent genes. It is important to understand the expression profile of the virulent genes. Quantitative RT-PCR is one of the preferred methods for determining different gene expressions. It is important to select the most stable gene as a reference in E...
September 23, 2016: Journal of Microbiology and Biotechnology
Anna Vavřínová, Michal Behuliak, Josef Zicha
OBJECTIVE: Catecholaminergic system plays an important role in blood pressure regulation and hypertension development. The available information concerning mRNA expression of catecholaminergic system genes in spontaneously hypertensive rats (SHR) are often contradictory. This might be due to various reference genes used as internal controls. We therefore searched for suitable reference genes for gene expression profiling in adrenal medulla and sympathetic ganglia of SHR and Wistar-Kyoto (WKY) rats, which would enable reliable comparison of mRNA expression of genes of catecholaminergic system between these strains...
September 2016: Journal of Hypertension
Bruno Paiva Dos Santos, Luciana Fraga da Costa Diesel, Lindolfo da Silva Meirelles, Nance Beyer Nardi, Melissa Camassola
This study was designed to (i) identify stable reference genes for the analysis of gene expression during in vitro differentiation of rat adipose stromal cells (rASCs), (ii) recommend stable genes for individual treatment conditions, and (iii) validate these genes by comparison with normalization results from stable and unstable reference genes. On the basis of a literature review, eight genes were selected: Actb, B2m, Hprt1, Ppia, Rplp0, Rpl13a, Rpl5, and Ywhaz. Genes were ranked according to their stability under different culture conditions as assessed using GenNorm, NormFinder, and RefFinder algorithms...
December 15, 2016: Gene
Pin Huan, Hongxia Wang, Baozhong Liu
The early development of mollusks exhibits important characteristics from the developmental and evolutionary perspective. With the increasing number of genome-wide studies, accurate analyses of quantitative gene expression during development are impeded by the lack of validated reference genes. To improve the situation, in this study, we analyzed the expression stability of seven candidate housekeeping genes during early development of the Pacific oyster Crassostrea gigas: actin, glyceraldehyde-3 phosphate-dehydrogenase (gapdh), α subunit of elongation factor 1 (elf1α), adp-ribosylation factor 1 (arf1), heterogeneous nuclear ribonucleoprotein q, ubiquitin-conjugating enzyme e2d2 and ribosomal protein s18...
August 31, 2016: Genes & Genetic Systems
Mariany Ashanty Morales, Bianca Marie Mendoza, Laura Corley Lavine, Mark Daniel Lavine, Douglas Bruce Walsh, Fang Zhu
Quantitative real-time PCR (qRT-PCR) is an extensively used, high-throughput method to analyze transcriptional expression of genes of interest. An appropriate normalization strategy with reliable reference genes is required for calculating gene expression across diverse experimental conditions. In this study, we aim to identify the most stable reference genes for expression studies of xenobiotic adaptation in Tetranychus urticae, an extremely polyphagous herbivore causing significant yield reduction of agriculture...
2016: International Journal of Biological Sciences
Kjersti Ringvoll Normann, Kristin Astrid Berland Øystese, Jens Petter Berg, Tove Lekva, Jon Berg-Johnsen, Jens Bollerslev, Nicoleta Cristina Olarescu
BACKGROUND: Real-time reverse transcription quantitative PCR (RT-qPCR) has become the method of choice for quantification of gene expression changes. The most important limitations of RT-qPCR are inappropriate data normalization and inconsistent data analyses. Pituitary adenomas are common tumours, and the appropriate interpretation of increasingly published data within this field is prevented by the lack of a proper selection and validation of stably expressed reference genes. AIM: To find and validate the optimal reference gene or gene combination for reliable RT-qPCR gene expression in both non-functioning (NFPA) and hormone secreting (GH and ACTH) pituitary adenomas...
December 5, 2016: Molecular and Cellular Endocrinology
Clemens L Bockmeyer, Karen Säuberlich, Juliane Wittig, Marc Eßer, Sebastian S Roeder, Udo Vester, Peter F Hoyer, Putri A Agustian, Philip Zeuschner, Kerstin Amann, Christoph Daniel, Jan U Becker
Small nucleolar RNAs (snoRNAs) have been used for normalization in glomerular microRNA (miRNA) quantification without confirmation of validity. Our aim was to identify glomerular reference miRNAs in IgA nephropathy. We compared miRNAs in human paraffin-embedded renal biopsies from patients with cellular-crescentic IgA-GN (n = 5; crescentic IgA-GN) and non-crescentic IgA-GN (n = 5; IgA-GN) to mild interstitial nephritis without glomerular abnormalities (controls, n = 5). Laser-microdissected glomeruli were used for expression profiling of 762 miRNAs by low-density TaqMan arrays (cards A and B)...
August 24, 2016: Scientific Reports
Li-Ting Deng, Yu-Ling Wu, Jun-Cheng Li, Kun-Xi OuYang, Mei-Mei Ding, Jun-Jie Zhang, Shu-Qi Li, Meng-Fei Lin, Han-Bin Chen, Xin-Sheng Hu, Xiao-Yang Chen
Moringa oleifera is a promising plant species for oil and forage, but its genetic improvement is limited. Our current breeding program in this species focuses on exploiting the functional genes associated with important agronomical traits. Here, we screened reliable reference genes for accurately quantifying the expression of target genes using the technique of real-time quantitative polymerase chain reaction (RT-qPCR) in M. oleifera. Eighteen candidate reference genes were selected from a transcriptome database, and their expression stabilities were examined in 90 samples collected from the pods in different developmental stages, various tissues, and the roots and leaves under different conditions (low or high temperature, sodium chloride (NaCl)- or polyethyleneglycol (PEG)- simulated water stress)...
2016: PloS One
Karen Staines, Ambalika Batra, William Mwangi, Helena J Maier, Steven Van Borm, John R Young, Mark Fife, Colin Butter
Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes, selected for the lowest variation in representative samples, can be used to control random technical variation. Reference gene assays must be reliable, have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts...
2016: PloS One
Qiusheng Kong, Lingyun Gao, Lei Cao, Yue Liu, Hameed Saba, Yuan Huang, Zhilong Bie
Melon (Cucumis melo L.) is an attractive model plant for investigating fruit development because of its morphological, physiological, and biochemical diversity. Quantification of gene expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR) with stably expressed reference genes for normalization can effectively elucidate the biological functions of genes that regulate fruit development. However, the reference genes for data normalization in melon fruits have not yet been systematically validated...
2016: Frontiers in Plant Science
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