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Ana Érika Inácio Gomes, Leonardo Prado Stuchi, Nathália Maria Gonçalves Siqueira, João Batista Henrique, Renato Vicentini, Marcelo Lima Ribeiro, Michelle Darrieux, Lúcio Fábio Caldas Ferraz
For reliable results, Reverse Transcription Quantitative real-time Polymerase Chain Reaction (RT-qPCR) analyses depend on stably expressed reference genes for data normalization purposes. Klebsiella pneumoniae is an opportunistic Gram-negative bacterium that has become a serious threat worldwide. Unfortunately, there is no consensus for an ideal reference gene for RT-qPCR data normalization on K. pneumoniae. In this study, the expression profile of eleven candidate reference genes was assessed in K. pneumoniae cells submitted to various experimental conditions, and the expression stability of these candidate genes was evaluated using statistical algorithms BestKeeper, NormFinder, geNorm, Delta CT and RefFinder...
June 13, 2018: Scientific Reports
Xiaoxiao Lu, Yongzhi Liu, Linchao Zhao, Yongnan Liu, Mingwen Zhao
This study aimed to identify suitable reference genes under three chemical inducers, methyl jasmonate (MeJA), salicylic acid (SA) and hydrogen peroxide (H2 O2 ) in Ganoderma lucidum. In this study, expression stabilities of 14 candidate reference genes had been validated. Four algorithms were used: geNorm, NormFinder, BestKeeper, and RefFinder. Our results showed that, in short time, UCE2 (ubiquitin conjugating enzyme) was the most stable gene both in MeJA and H2 O2 treatments, ACTIN (beta-actin) was the most suitable reference gene for SA treatment...
June 12, 2018: World Journal of Microbiology & Biotechnology
Xuelin Zhao, Jianping Fu, Liting Jiang, Weiwei Zhang, Yina Shao, Chunhua Jin, Jinbo Xiong, Chenghua Li
Quantitative real-time PCR (qRT-PCR) is a standard method to measure gene expression in function exploring. Accurate and reproducible data of qRT-PCR requires appropriate reference genes, which are stably expressed under different experimental conditions. However, no housekeeping genes were validated as internal controls for qRT-PCR in Sinonovacula constricta. In this study, we classified the transcriptome data of two tissues for Vibrio infection and Cd2+ stress into ten clusters based on the gene expression patterns...
June 2018: Genes & Genomics
Cheng Qu, Ran Wang, Wunan Che, Xun Zhu, Fengqi Li, Chen Luo
Harmonia axyridis (Coleoptera: Coccinellidae) is a polyphagous insect that is an important biological agent used to control agricultural and forestry pests. The role of functional genes in H. axyridis based on quantitative real-time PCR (qRT-PCR) is increasingly well understood to investigate biology, physiology, feeding behavior and the role of important genes in physiological processes. Quantitative real-time PCR (qRT-PCR) is a powerful and reliable technique to quantify gene expression. Using qRT-PCR, expression levels of target genes are determined based on the levels of internal reference genes; therefore, reference genes need to be stably expressed under specific experimental conditions...
2018: PloS One
Wenxian Liang, Xiaoxing Zou, Rebeca Carballar-Lejarazú, Lingjiao Wu, Weihong Sun, Xueyuan Yuan, Songqing Wu, Pengfei Li, Hui Ding, Lin Ni, Wei Huang, Shuangquan Zou
Background: Quantitative real-time reverse transcription-polymerase chain reaction has been widely used in gene expression analysis, however, to have reliable and accurate results, reference genes are necessary to normalize gene expression under different experimental conditions. Several reliable reference genes have been reported in plants of Traditional Chinese Medicine, but none have been identified for Euscaphis konishii Hayata. Results: In this study, 12 candidate reference genes, including 3 common housekeeping genes and 9 novel genes based on E...
2018: Plant Methods
Julian Geiger, Louise T Dalgaard
The presence of noncoding RNAs, such as microRNAs (miRNAs), in mitochondria has been reported by several studies. The biological roles and functions of these mitochondrial miRNAs ("mitomiRs") have not been sufficiently characterized, but the mitochondrial localization of miRNAs has recently gained significance due to modified mitomiR-populations in certain states of diseases. Here, we describe the isolation and analysis of mitochondrial RNAs from rat liver tissue and HepG2 cells. The principle of the analysis is to prepare mitochondria by differential centrifugation...
2018: Methods in Molecular Biology
Federica Banfi, Alessandra Colombini, Carlotta Perucca Orfei, Valentina Parazzi, Enrico Ragni
The molecular profile of human mesenchymal stem cells (MSCs) have emerged as a key factor in defining their identity. Nevertheless, the effect of fetal bovine serum (FBS) batches or origin on MSC molecular signature has been neglected. In this frame, chemical fingerprint of FBS batches from unrelated countries showed strong correlation between chemical composition and country of origin. Thus, the aim of this study was to evaluate in stem cells isolated from bone marrow (BMMSCs) and umbilical cord-blood (CBMSCs) the effects of independently collected FBS batches on both twelve commonly used reference genes (RGs) and a selected panel of thirty-eight genes crucial for MSC definition in both research and clinical settings...
May 26, 2018: Stem Cell Reviews
Shuai Peng, Longxiang Liu, Hongyu Zhao, Hua Wang, Hua Li
The powerful Quantitative real-time PCR (RT-qPCR) was widely used to assess gene expression levels, which requires the optimal reference genes used for normalization. Oenococcus oeni ( O. oeni ), as the one of most important microorganisms in wine industry and the most resistant lactic acid bacteria (LAB) species to ethanol, has not been investigated regarding the selection of stable reference genes for RT-qPCR normalization under ethanol stress conditions. In this study, nine candidate reference genes ( proC, dnaG, rpoA, ldhD, ddlA, rrs, gyrA, gyrB , and dpoIII ) were analyzed to determine the most stable reference genes for RT-qPCR in O...
2018: Frontiers in Microbiology
Marcelina E Janik, Sabina Szwed, Paweł Grzmil, Radosław Kaczmarek, Marcin Czerwiński, Dorota Hoja-Łukowicz
The objective of this study was to identify a normalizer or combination of normalizers for quantitative evaluation of the expression of a target gene of interest during melanoma progression. Adult melanocytes, uveal primary melanoma cells and cutaneous primary and metastatic melanoma cells were used to construct a panel of 14 experimental models reflecting cancer promotion and progression. Hypoxanthine phosphoribosyltransferase 1 (HPRT1), glucuronidase beta (GUSB), ribosomal protein S23 (RPS23), phosphoglycerate kinase 1 (PGK1) and small nuclear ribonucleoprotein polypeptide A (SRNPA) were chosen as candidate housekeeping genes...
May 17, 2018: International Journal of Biochemistry & Cell Biology
Jason G Walling, Leslie A Zalapa, Marcus A Vinje
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a popular method for measuring transcript abundance. The most commonly used method of interpretation is relative quantification and thus necessitates the use of normalization controls (i.e. reference genes) to standardize transcript abundance. The most popular gene targets for RT-qPCR are housekeeping genes because they are thought to maintain a static transcript level among a variety of samples. However, more recent studies have shown, several housekeeping genes are not reliably stable...
2018: PloS One
Lusheng Xin, Bowen Huang, Changming Bai, Chongming Wang
Ostreid herpesvirus-1 (OsHV-1) presents interspecies transmission among bivalves. Recently, events of mass mortalities of ark clams (Scapharca broughtonii) infected with OsHV-1 have been recorded. To accurately assess the gene responding patterns of ark clams post OsHV-1 infection, constant stable housekeeping genes (HKGs) are needed as internal control to normalize raw mRNA expression data. In this study, ten candidate HKGs were selected, including 18S rRNA (18S), beta-actin (ACT), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), NADH dehydrogenase subunit (NADH), Elongation factor-1a (EF-1a), Elongation factor-1β (EF-1β), Elongation factor-1γ (EF-1γ), Ribosomal protein L7 (RL7), Ribosomal protein L15 (RL15) and Ribosomal protein S18 (S18)...
April 30, 2018: Journal of Invertebrate Pathology
Benshui Shu, Jingjing Zhang, Gaofeng Cui, Ranran Sun, Veeran Sethuraman, Xin Yi, Guohua Zhong
Azadirachtin is an efficient and broad-spectrum botanical insecticide against more than 150 kinds of agricultural pests with the effects of mortality, antifeedant and growth regulation. Real-time quantitative polymerase chain reaction (RT-qPCR) could be one of the powerful tools to analyze the gene expression level and investigate the mechanism of azadirachtin at transcriptional level, however, the ideal reference genes are needed to normalize the expression profiling of target genes. In this present study, the fragments of eight candidate reference genes were cloned and identified from the pest Spodoptera litura ...
2018: Frontiers in Physiology
Bo Wang, Huihui Du, Zhengpei Yao, Cai Ren, Li Ma, Jiao Wang, Hua Zhang, Hao Ma
Haloxylon ammodendron plays an important role in maintaining the structure and function of the entire ecosystem where it grows. No suitable reference genes have been reported in H. ammodendron plants to date. In this study, a total of 8 reference genes ( 18S , ACT1 , ACT7 , UBC18 , TUA5 , GAPDH , EF - 1α and UBQ10 ) were selected from the available trancriptome database, and the expression stability of these 8 candidate genes was validated under different abiotic stress with three different statistical algorithms (geNorm, NormFinder and BestKeeper)...
May 2018: Physiology and Molecular Biology of Plants: An International Journal of Functional Plant Biology
Viviane S Moreira, Virgínia L F Soares, Raner J S Silva, Aurizangela O Sousa, Wagner C Otoni, Marcio G C Costa
Bixa orellana L., popularly known as annatto, produces several secondary metabolites of pharmaceutical and industrial interest, including bixin, whose molecular basis of biosynthesis remain to be determined. Gene expression analysis by quantitative real-time PCR (qPCR) is an important tool to advance such knowledge. However, correct interpretation of qPCR data requires the use of suitable reference genes in order to reduce experimental variations. In the present study, we have selected four different candidates for reference genes in B...
May 2018: Physiology and Molecular Biology of Plants: An International Journal of Functional Plant Biology
Chunyan Tu, Tieshuai Du, Chengchen Shao, Zengjia Liu, Liliang Li, Yiwen Shen
The precise estimation of postmortem interval (PMI) is a critical step in death investigation of forensic cases. Detecting the degradation of RNA in tissues by real time quantitative polymerase chain reaction (RT-qPCR) technology provides a new theoretical basis for estimation of PMI. However, most commonly used reference genes degrade over time, while previous studies seldom consider this when selecting suitable reference genes for the estimation of PMI. Studies have shown microRNAs (miRNAs) are very stable and circular RNAs (circRNAs) have recently emerged as a novel class of RNAs with high stability...
April 24, 2018: Forensic Science, Medicine, and Pathology
Marco Viganò, Carlotta Perucca Orfei, Laura de Girolamo, John R Pearson, Enrico Ragni, Paola De Luca, Alessandra Colombini
The use of biochemical inducers of mesenchymal stem cell (MSC) differentiation into tenogenic lineage represents an investigated aspect of tendon disorder treatment. Bone morphogenetic protein 12 (BMP-12) is a widely studied factor, representing along with ascorbic acid (AA) and basic fibroblast growth factor (bFGF) one of the most promising stimulus in this context so far. Quantitative gene expression of specific tenogenic marker is commonly used to assess the efficacy of these supplements. Nevertheless, the reliability of these data is strongly associated with the choice of stable housekeeping genes...
May 16, 2018: Tissue Engineering. Part C, Methods
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)...
May 2018: Journal of Microbiological Methods
Qian Yang, Jinyao Li, Jing Shen, Yufang Xu, Hongjie Liu, Wei Deng, Xuefeng Li, Mingqi Zheng
Descurainia sophia is one of the most notorious broadleaf weeds in China and has evolved extremely high resistance to acetolactate synthase (ALS)-inhibiting herbicide tribenuron-methyl. The target-site resistance due to ALS gene mutations was well-known, 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...
May 2, 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
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