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Drew M DeLorenzo, Tae Seok Moon
Rhodococcus opacus PD630 is a gram-positive bacterium with promising attributes for the conversion of lignin into valuable fuels and chemicals. To develop an organism as a cellular factory, it is necessary to have a deep understanding of its metabolism and any heterologous pathways being expressed. For the purpose of quantifying gene transcription, reverse transcription quantitative PCR (RT-qPCR) is the gold standard due to its sensitivity and reproducibility. However, RT-qPCR requires the use of reference genes whose expression is stable across distinct growth or treatment conditions to normalize the results...
April 16, 2018: Scientific Reports
Liang Yang, Juanni Chen, Ying Liu, Shuting Zhang, Shili Li, Wei Ding
Quantitative real-time reverse transcriptase PCR (qRT-PCR) has become the method choice for quantification of gene expression changes, however, the accuracy of the method depends on the stability of reference genes. Ralstonia pseudosolanacearum (R. pseudosolanacearum) is an important plant pathogen, infecting >450 plant species and causing bacterial wilt. In order to identify stable reference genes in R. pseudosolanacearum CQPS-1 under different environment stresses. We used five tools (△Ct method, GeNorm, NormFinder, BestKeeper, and RefFinder) to evaluate the stability of seven candidate reference genes including phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 16S ribosomal RNA (16S), cell division protein ftsZ (ftsZ), DNA gyrase subunit A (gyrA), Ribosomal protein L13 (rplM), and phosphoserine aminotransferase (serC) under biotic (growth phases) and abiotic stress (temperature, hydroxycoumarins, nutrition)...
April 10, 2018: Journal of Microbiological Methods
Qian Yang, Jinyao Li, Jing Shen, Yufang Xu, Hongjie Liu, Wei Deng, Xuefeng Li, Mingqi Zheng
D. sophia is one of the most notorious broadleaf weed in China, and has evolved extremely high resistance to ALS-inhibiting herbicide tribenuron-methyl. The target-site resistance due to ALS gene mutations was known well, while the non-target-site resistance is not yet well-characterized. Metabolic resistance, which is conferred by enhanced rates of herbicide metabolism, is the most important NTSR. To explore the mechanism of metabolic resistance underlying resistant (R) D. sophia plants, tribenuron-methyl uptake and metabolism levels, qPCR reference gene stability, and candidate P450 genes expression patterns were investigated...
April 13, 2018: Journal of Agricultural and Food Chemistry
Katsushige Inada, Yasushi Okoshi, Yukiko Cho-Isoda, Shingo Ishiguro, Hisashi Suzuki, Akinori Oki, Yoshio Tamaki, Toru Shimazui, Hitoaki Saito, Mitsuo Hori, Tatsuo Iijima, Hiroshi Kojima
Lymph node metastasis is one of the most important factors for tumor dissemination. Quantifying microRNA (miRNA) expression using real-time PCR in formalin-fixed, paraffin-embedded (FFPE) lymph node can provide valuable information regarding the biological research for cancer metastasis. However, a universal endogenous reference gene has not been identified in FFPE lymph node. This study aimed to identify suitable endogenous reference genes for miRNA expression analysis in FFPE lymph node. FFPE lymph nodes were obtained from 41 metastatic cancer and from 16 non-cancerous tissues...
April 12, 2018: Scientific Reports
Yanchun You, Miao Xie, Liette Vasseur, Minsheng You
Gene expression analysis provides important clues regarding gene functions, and quantitative real-time PCR (qRT-PCR) is a widely-used method for gene expression studies. Reference genes are essential for normalizing and accurately assessing gene expressions. In the present study, 16 candidate reference genes (<i>ACTB</i>, <i>CyPA</i>, <i>EF1-α</i>, <i>GAPDH</i>, <i>HSP90</i>, <i>NDPk</i>, <i>RPL13a</i>, <i>RPL18</i>, <i>RPL19</i>, <i>RPL32</i>, <i>RPL4</i>, <i>RPL8</i>, <i>RPS13</i>, <i>RPS4</i>, <i>α-TUB</i> and <i>β-TUB</i>) of <i>Plutella xylostella</i> were selected to evaluate their gene expression stability across different experimental conditions using five statistical algorithms (geNorm, NormFinder, Delta Ct method, BestKeeper and RefFinder)...
April 5, 2018: Genome Génome / Conseil National de Recherches Canada
Hong-Bo Li, Chang-Geng Dai, Chang-Rong Zhang, Yong-Fu He, Hai-Yan Ran, Shi-Hong Chen
The oriental armyworm, Mythimna separata, is a major insect pest in China and other Asian countries. Unfortunately, suitable reference genes for quantitative real-time PCR (qRT-PCR) have not been previously identified in M. separata for normalizing target gene expression. In this study, we evaluated the expression stability of eight candidate genes (18S, ACT, EF1-α, GAPDH, RPS7, RPS13, RPL32 and TUB) in M. separata using the comparative ΔCt method, BestKeeper, Normfinder geNorm and ReFinder, a comprehensive software platform...
2018: PloS One
Fernando Vera Hernández Pedro, Marcelino Martínez Núñez, Magali Ruiz Rivas, Ramiro Eduardo Vázquez Portillo, Martha Dolores Bibbins Martínez, Silvia Luna Suárez, Flor de Fátima Rosas Cárdenas
Studies of gene expression are very important for the identification of genes that participate in different biological processes. Currently, reverse transcription quantitative real-time PCR (RT-qPCR) is a high-throughput, sensitive and widely used method forgene expression analysis. Nevertheless, RT-qPCR requires a precise normalization of data to avoid the misinterpretation of experimental data. In this sense, the selection of reference genes is critical for gene expression analysis. At this time, several studies are focused on the selection of reference genes in several plants...
March 30, 2018: Plant Biology
Xitong Fei, Qianqian Shi, Tuxi Yang, Zhaoxue Fei, Anzhi Wei
Real-time reverse transcription quantitative PCR has become a common method for studying gene expression, however, the optimal selection of stable reference genes is a prerequisite for obtaining accurate quantification of transcript abundance. Suitable reference genes for RT-qPCR have not yet been identified for Chinese prickly ash ( Zanthoxylum bungeanum Maxim.). Chinese prickly ash is the source of an important food seasoning in China. In recent years, Chinese prickly ash has also been developed as a medicinal plant...
March 30, 2018: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Xiao-Lin Wang, Jiao-Jiao Ji, Jian-Ping Gao
GAPDH(glyceraldehyde-3-phosphate dehydrogenase) gene is a key enzyme gene in carbohydrate metabolism and always used as reference gene. To clarify and complete the biosynthetic pathway of polysaccharide, the GAPDH gene in Codonopsis pilosula, named CpGAPDH, was cloned according to the transcriptome of pilosula, using the GAPDH gene in potato as query. The CpGAPDH contained a 1 014 bp open reading frame(ORF) and encoded a protein with 337 amino acids. Bioinformatic analysis clearly suggested that CpGAPDH shared high similarity with GAPDH among other plants, and had the closest relatives to potato and danshen...
February 2018: Zhongguo Zhong Yao za Zhi, Zhongguo Zhongyao Zazhi, China Journal of Chinese Materia Medica
Duc Quan Nguyen, Andrew L Eamens, Christopher P L Grof
Background: Quantitative real-time polymerase chain reaction (RT-qPCR) is the key platform for the quantitative analysis of gene expression in a wide range of experimental systems and conditions. However, the accuracy and reproducibility of gene expression quantification via RT-qPCR is entirely dependent on the identification of reliable reference genes for data normalisation. Green foxtail ( Setaria viridis ) has recently been proposed as a potential experimental model for the study of C4 photosynthesis and is closely related to many economically important crop species of the Panicoideae subfamily of grasses, including Zea mays (maize), Sorghum bicolor (sorghum) and Sacchurum officinarum (sugarcane)...
2018: Plant Methods
Tangchun Zheng, Zhilin Chen, Yiqian Ju, Han Zhang, Ming Cai, Huitang Pan, Qixiang Zhang
Quantitative real-time polymerase chain reaction (qRT-PCR) is a prevalent method for gene expression analysis, depending on the stability of the reference genes for data normalization. Lagerstroemia indica and L. speciosa are popular ornamental plants which are famous for the long flowering period. However, no systematic studies on reference genes in Lagerstroemia have yet been conducted. In the present study, we selected nine candidate reference genes (GAPDH, TUA, TUB, 18S, RPII, EF-1α, ATC, EIF5A and CYP) and evaluated their expression stability in different tissues during floral development of L...
2018: PloS One
Genhong Wang, Yanfei Chen, Xiaoying Zhang, Bingchuan Bai, Hao Yan, Daoyuan Qin, Qingyou Xia
The silkworm, Bombyx mori, is one of the world's most economically important insect. Surveying variations in gene expression among multiple tissue/organ samples will provide clues for gene function assignments and will be helpful for identifying genes related to economic traits or specific cellular processes. To ensure their accuracy, commonly used gene expression quantification methods require a set of stable reference genes for data normalization. In this study, 24 candidate reference genes were assessed in 10 tissue/organ samples of day 3 fifth-instar B...
March 23, 2018: Archives of Insect Biochemistry and Physiology
Najat Dzaki, Ghows Azzam
Members of the Aedes genus of mosquitoes are widely recognized as vectors of viral diseases. Ae.albopictus is its most invasive species, and are known to carry viruses such as Dengue, Chikugunya and Zika. Its emerging importance puts Ae.albopictus on the forefront of genetic interaction and evolution studies. However, a panel of suitable reference genes specific for this insect is as of now undescribed. Nine reference genes, namely ACT, eEF1-γ, eIF2α, PP2A, RPL32, RPS17, PGK1, ILK and STK were evaluated. Expression patterns of the candidate reference genes were observed in a total of seventeen sample types, separated by stage of development and age...
2018: PloS One
Chaoqiong Liang, Jianjun Hao, Yan Meng, Laixin Luo, Jianqiang Li
Cucumber green mottle mosaic virus (CGMMV) is an economically important pathogen and causes significant reduction of both yield and quality of cucumber (Cucumis sativus). Currently, there were no satisfied strategies for controlling the disease. A better understanding of microRNA (miRNA) expression related to the regulation of plant-virus interactions and virus resistance would be of great assistance when developing control strategies for CGMMV. However, accurate expression analysis is highly dependent on robust and reliable reference gene used as an internal control for normalization of miRNA expression...
2018: PloS One
Fanwei Dai, Xiting Zhao, Cuiming Tang, Zhenjiang Wang, Zheshi Kuang, Zhiyi Li, Jing Huang, Guoqing Luo
Mulberry (Morus alba L.) is an important economic tree species in many countries. Quantitative real time PCR (qRT-PCR) has become a widely used method for gene expression studies in plants. A suitable reference gene is essential to ensure accurate and reliable results for qRT-PCR analyses. However, no reports describing the selection of reference genes have been published for mulberry. In this work, we evaluated the stability of twenty candidate reference genes in different plant tissues and under different stress conditions by qRT-PCR in mulberry using algorithms in two programs-geNorm and NormFinder...
2018: PloS One
Meng Luo, Zhen Gao, Hui Li, Qin Li, Caixi Zhang, Wenping Xu, Shiren Song, Chao Ma, Shiping Wang
Grapevine is among the fruit crops with high economic value, and because of the economic losses caused by abiotic stresses, the stress resistance of Vitis vinifera has become an increasingly important research area. Among the mechanisms responding to environmental stresses, the role of miRNA has received much attention recently. qRT-PCR is a powerful method for miRNA quantitation, but the accuracy of the method strongly depends on the appropriate reference genes. To determine the most suitable reference genes for grapevine miRNA qRT-PCR, 15 genes were chosen as candidate reference genes...
March 13, 2018: Scientific Reports
Clemens L Bockmeyer, Juliane Wittig, Karen Säuberlich, Philipp Selhausen, Marc Eßer, Philip Zeuschner, Friedrich Modde, Kerstin Amann, Christoph Daniel
BACKGROUND: Glomeruli are excellent pre-determined natural structures for laser micro-dissection. Compartment-specific glomerular gene expression analysis of formalin-fixed paraffin-embedded renal biopsies could improve research applications. The major challenge for such studies is to obtain good-quality RNA from small amounts of starting material, as applicable for the analysis of glomerular compartments. In this work, we provide data and recommendations for an optimized workflow of glomerular mRNA analysis...
March 13, 2018: BMC Molecular Biology
Jinxing Wang, Manzar Abbas, Yanzhong Wen, Dongsheng Niu, Ling Wang, Yuhan Sun, Yun Li
Black locust (Robinia pseudoacacia L.) is an easy to raise, fast growing, medium-sized deciduous tree species highly tolerant to harsh eco-conditions, i.e., drought and harsh winters, and it is widely adaptable to sandy, loamy, and marshy soils. The basis for this adaptability remains to be investigated at the transcriptomic level using real-time quantitative PCR (qPCR). Selection of a reliable gene for the normalization of qPCR data is important for obtaining accurate results in gene expression. The goal of this study was to identify an appropriate reference gene from 12 candidate genes for gene expression analysis in black locust exposed to various stressors such as abscisic acid (ABA), NaCl, polyethylene glycol (PEG) and varying temperatures...
2018: PloS One
Sheng Wang, Jianqing Wang, Xiongwen Lv
Investigations of hepatic gene expression are crucial for determining the molecular factors involved in acute alcoholic liver injury. The results of liver molecular investigations may reveal etiologically important genomic alterations. Therefore, it is necessary to normalize gene expression data to identify stable genes, which may be used as a reference under different experimental conditions. The aim of the present study was to apply reverse transcription‑quantitative polymerase chain reaction analysis and use analysis software to investigate the expression stability of candidate reference genes in hepatic tissues from mice with acute alcoholic liver injury...
March 1, 2018: International Journal of Molecular Medicine
Ramneek Kaur, Monika Sodhi, Ankita Sharma, Vijay Lakshmi Sharma, Preeti Verma, Shelesh Kumar Swami, Parvesh Kumari, Manishi Mukesh
Selection of reference genes has become an integral step in any real time quantitative PCR (RT-qPCR) based expression studies. The importance of this study stems from the fact that riverine buffaloes are major dairy species of Indian sub-continent and the information generated here will be of great interest to the investigators engaged in functional genomic studies of this important livestock species. In this study, an effort was made to evaluate a panel of 10 candidate reference genes (glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta- actin (ACTB), ubiquitously expressed transcript (UXT), ribosomal protein S15 (RPS15), ribosomal protein L-4 (RPL4), ribosomal protein S9 (RPS9), ribosomal protein S23 (RPS23), hydroxymethylbilane synthase (HMBS), β2 Microglobulin (β2M) and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) across 12 tissues (mammary gland, kidney, spleen, liver, heart, intestine, ovary, lung, muscle, brain, subcutaneous fat and testis) of riverine buffaloes...
2018: PloS One
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