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Xiaowei Yang, Huipeng Pan, Ling Yuan, Xuguo Zhou
Harmonia axyridis is a voracious predator, a biological control agent, and one of the world most invasive insect species. The advent of next-generation sequencing platforms has propelled entomological research into the genomics and post-genomics era. Real-time quantitative PCR (RT-qPCR), a primary tool for gene expression analysis, is a core technique governs the genomic research. The selection of internal reference genes, however, can significantly impact the interpretation of RT-qPCR results. The overall goal of this study is to identify the reference genes in the highly invasive H...
February 9, 2018: Scientific Reports
XinYue Wang, Feng Peng, Guibin Dong, Yang Sun, Xiaofeng Dai, Yankun Yang, Xiuxia Liu, Zhonghu Bai
Real-time quantitative PCR (qRT-PCR) is a fast and efficient technology for detecting gene expression levels in the study of the Corynebacterium glutamicum protein expression system, but it requires normalization to ensure the reliability of the results obtained. We selected 13 genes from the commonly used housekeeping genes and from transcriptome data as candidate reference genes. The Ct values of the 13 genes were obtained by qRT-PCR at different fermentation stages and under three stress conditions (temperature, acid, and salt)...
February 6, 2018: FEMS Microbiology Letters
S A Sheshadri, M J Nishanth, V Yamine, Bindu Simon
The role of Melatonin in influencing diverse genes in plants has gained momentum in recent years and many reports have employed qRT-PCR for their quantification. Relative quantification of gene expression relies on accurate normalization of qRT-PCR data against a stably-expressing internal reference-gene. Although researchers have been using commonly available reference-genes to assess Melatonin-induced gene expression, but to-date, there have been no attempts to validate the reference-gene stability under Melatonin-supplementation in planta...
February 5, 2018: Scientific Reports
Yingbo Kang, Zhuomin Wu, De Cai, Binger Lu
BACKGROUND: Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a critical tool for evaluating the levels of mRNA transcribed from genes. Reliable RT-qPCR results largely depend on normalization to suitable reference genes. Middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation/reoxygenation (OGD/R) are models that are commonly used to study ischemic stroke. However, the proper reference genes for RNA analysis in these two models have not yet been determined...
February 1, 2018: BMC Neuroscience
Izabela Ruduś, Jan Kępczyński
Molecular studies of primary and secondary dormancy in Avena fatua L., a serious weed of cereal and other crops, are intended to reveal the species-specific details of underlying molecular mechanisms which in turn may be useable in weed management. Among others, quantitative real-time PCR (RT-qPCR) data of comparative gene expression analysis may give some insight into the involvement of particular wild oat genes in dormancy release, maintenance or induction by unfavorable conditions. To assure obtaining biologically significant results using this method, the expression stability of selected candidate reference genes in different data subsets was evaluated using four statistical algorithms i...
2018: PloS One
Hui Zhao, Teng-Fei Ma, Jie Lin, Lin-Lin Liu, Wei-Jie Sun, Li-Xia Guo, Si-Qi Wang, Newton O Otecko, Ya-Ping Zhang
RT-qPCR offers high sensitivity, for accurate interpretations of qPCR results however, normalisation using suitable reference genes is fundamental. Androgens can regulate transcriptional expression including reference gene expression in prostate cancer. In this study, we evaluated ten mRNA and six non-protein coding RNA reference genes in five prostate cell lines under varied dihydrotestosterone (DHT) treatments. We validated the effects of DHT-treatments using media containing charcoal-stripped serum prior to DHT stimulation on the test samples by Western blot experiments...
January 31, 2018: Scientific Reports
Tsuguhisa Nakayama, Naoko Okada, Mamoru Yoshikawa, Daiya Asaka, Akihito Kuboki, Hiromi Kojima, Yasuhiro Tanaka, Shin-Ichi Haruna
Reverse transcription-quantitative polymerase chain reaction is a valuable and reliable method for gene quantification. Target gene expression is usually quantified by normalization using reference genes (RGs), and accurate normalization is critical for producing reliable data. However, stable RGs in nasal polyps and sinonasal tissues from patients with chronic rhinosinusitis (CRS) have not been well investigated. Here, we used a two-stage study design to identify stable RGs. We assessed the stability of 15 commonly used candidate RGs using five programs-geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder...
January 25, 2018: Scientific Reports
Anita Ciesielska, Paweł Stączek
Dermatophytes are the group of filamentous fungi infecting keratinized structures such as skin, hair, and nails. Knowledge about genes and molecular mechanisms responsible for pathogenicity, as well as other biological properties of Microsporum canis is still relatively poor. The qRT-PCR is a reliable technique for quantifying gene expression across various biological processes, and choosing a set of suitable reference genes to normalize the expression data is a crucial step of this technique. We investigated the suitability of nine candidate reference genes: β-act, β-tub, adp-rf, ef1-α, sdha, rpl2, mbp1, psm1, and rGTPa for gene expression analysis in the dermatophyte M...
January 19, 2018: Scientific Reports
Qiuxu Liu, Xiao Qi, Haidong Yan, Linkai Huang, Gang Nie, Xinquan Zhang
To select the most stable reference genes in annual ryegrass (Lolium multiflorum), we studied annual ryegrass leaf tissues exposed to various abiotic stresses by qRT-PCR and selected 11 candidate reference genes, i.e., 18S rRNA, E2, GAPDH, eIF4A, HIS3, SAMDC, TBP-1, Unigene71, Unigene77, Unigene755, and Unigene14912. We then used GeNorm, NormFinder, and BestKeeper to analyze the expression stability of these 11 genes, and used RefFinder to comprehensively rank genes according to stability. Under different stress conditions, the most suitable reference genes for studies of leaf tissues of annual ryegrass were different...
January 16, 2018: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Zayneb Chaâbene, Agnieszka Rorat, Imen Rekik Hakim, Fabien Bernard, Grubb C Douglas, Amine Elleuch, Franck Vandenbulcke, Hafedh Mejdoub
Phytochelatin synthase and metallothionein gene expressions were monitored via qPCR in order to investigate the molecular mechanisms involved in Cd and Cr detoxification in date palm (Phoenix dactylifera). A specific reference gene validation procedure using BestKeeper, NormFinder and geNorm programs allowed selection of the three most stable reference genes in a context of Cd or Cr contamination among six reference gene candidates, namely elongation factor α1, actin, aldehyde dehydrogenase, SAND family, tubulin 6 and TaTa box binding protein...
December 28, 2017: Chemosphere
Satnam Singh, Mridula Gupta, Suneet Pandher, Gurmeet Kaur, Pankaj Rathore, Subba Reddy Palli
Amrasca biguttula biguttula (Ishida) commonly known as cotton leafhopper is a severe pest of cotton and okra. Not much is known on this insect at molecular level due to lack of genomic and transcriptomic data. To prepare for functional genomic studies in this insect, we evaluated 15 common housekeeping genes (Tub, B-Tub, EF alpha, GADPH, UbiCF, RP13, Ubiq, G3PD, VATPase, Actin, 18s, 28s, TATA, ETF, SOD and Cytolytic actin) during different developmental stages and under starvation stress. We selected early (1st and 2nd), late (3rd and 4th) stage nymphs and adults for identification of stable housekeeping genes using geNorm, NormFinder, BestKeeper and RefFinder software...
2018: PloS One
Bilal B Mughal, Michelle Leemans, Petra Spirhanzlova, Barbara Demeneix, Jean-Baptiste Fini
Reference genes are essential for gene expression analysis when using real-time quantitative PCR (RT-qPCR). Xenopus laevis is a popular amphibian model for studying vertebrate embryogenesis and development. Further, X. laevis is ideal for studying thyroid signaling due to its thyroid dependent metamorphosis, a stage comparable to birth in humans. When using PCR based studies, a primary concern is the choice of reference genes. Commonly used references are eef1a1, odc1, rpl8, and actnB, although there is a lack of ad hoc reference genes for X...
January 11, 2018: Scientific Reports
Xiaohua Xia, Weiran Huo, Ruyan Wan, Xiaopei Xia, Qiyan Du, Zhongjie Chang
Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a well-known method to quantify gene expression by comparing with the reference genes. Generally, housekeeping genes were set as references, as for their stable expression in varying conditions. Here, we try to evaluate few of such genes to identify suitable housekeeping genes as references for qRT-PCR analysis of gene expression in Misgurnus anguillicaudatus. This study evaluated the expression of four commonly used housekeeping genes, i...
December 2017: Journal of Genetics
Jian Liu, Shengxiong Huang, Xiangli Niu, Danyang Chen, Qiang Chen, Li Tian, FangMing Xiao, Yongsheng Liu
The appropriate reference genes are important and essential for reliable results of transcript normalization in real-time qRT-PCR. In the current study, we identified 1203 stably expressed genes from 35,286 genes' expression profiles in developmental fruits of Actinidia chinensis. We manually selected six candidate genes and assessed their expression levels, using two sets of fruit samples of A. chinensis: flesh fruits at four developmental stages and post-harvested fruits. The expression stability of these six genes was assessed by three independent algorithms: geNorm, NormFinder, and BestKeeper...
December 11, 2017: Gene
Joey St-Pierre, Jean-Charles Grégoire, Cathy Vaillancourt
Normalization with proper reference genes is a crucial step in obtaining accurate mRNA expression levels in RT-qPCR experiments. GeNorm and NormFinder are two commonly used software packages that help in selecting the best reference genes, based on their expression stability. However, GeNorm does not take into account a group variable, such as sample sex, in its calculation. We demonstrate a simple calculation step to assess the variability of such parameters by multiplying the GeNorm M value with the difference of Cq values between groups...
December 5, 2017: Scientific Reports
Yung-Yi C Mosley, Harm HogenEsch
BACKGROUND: The quantification of gene expression is an important tool in the evaluation of the immune response to vaccines. Reliable reference genes for gene expression studies in mouse draining lymph nodes after vaccination have not been reported. RESULTS: The utility of seven potential reference genes was investigated using commercially available Taq-man primer/probe mixes. Results were evaluated with RefFinder, a web-based program including multiple algorithm methods such as geNorm, NormFinder, BestKeeper and the comparative delta-Ct...
December 6, 2017: BMC Research Notes
Nikoletta A Nagy, Zoltán Németh, Edit Juhász, Szilárd Póliska, Rita Rácz, András Kosztolányi, Zoltán Barta
Hormones play an important role in the regulation of physiological, developmental and behavioural processes. Many of these mechanisms in insects, however, are still not well understood. One way to investigate hormonal regulation is to analyse gene expression patterns of hormones and their receptors by real-time quantitative polymerase chain reaction (RT-qPCR). This method, however, requires stably expressed reference genes for normalisation. In the present study, we evaluated 11 candidate housekeeping genes as reference genes in samples of Lethrus apterus, an earth-boring beetle with biparental care, collected from a natural population...
2017: PeerJ
Pouya Dini, Shavahn C Loux, Kirsten E Scoggin, Alejandro Esteller-Vico, Edward L Squires, Mats H T Troedsson, Peter Daels, Barry A Ball
MicroRNAs (miRNAs) have important posttranscriptional regulatory abilities, and there is considerable interest in evaluating their expression patterns in different pathophysiological states. The most common method of quantifying miRNA expression is quantitative reverse transcription PCR; however, the identification of tissue-specific and species-specific reference miRNA is a prerequisite for miRNA expression analysis. Currently, no reference genes have been described for evaluating miRNA expression in equine serum and chorioallantoic membrane (CAM) during pregnancy...
December 2, 2017: Molecular Biotechnology
Xue-Ke Gao, Shuai Zhang, Jun-Yu Luo, Chun-Yi Wang, Li-Min Lü, Li-Juan Zhang, Xiang-Zhen Zhu, Li Wang, Jin-Jie Cui
Reference genes have been utilized in estimating gene expression levels using quantitative reverse transcriptase-quantitative polymerase chain reaction (qRT-PCR) analysis. Aphidius gifuensis Ashmaed is one of the most widely used biological control agents for aphids. The biological properties of this species have been studied in detail, and current investigations are focused on elucidating the regulatory mechanisms in its host However, the appropriate reference genes for target gene expression studies have not been identified...
2017: PloS One
L Scarabel, A Milani, S Panozzo, A Rasori
Resistance to 2,4-D (2,4-diclorophenoxyacetic acid) herbicide is increasing in various dicotyledonous weed species, including Papaver rhoeas, a weed infesting Southern European wheat crops. Non-target-site resistance to this herbicide is governed by a range of genes involved in herbicide stress response. To enable reliable measurement of gene expression levels in herbicide-resistant and susceptible plants it is necessary to normalize qPCR data using internal control genes with stable expression. In an attempt to find the best reference genes, the stability of seven candidate reference genes was assessed in plants resistant and susceptible to 2,4-D, subjected or not to herbicide stress...
November 2017: Pesticide Biochemistry and Physiology
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