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Validation of Suitable Reference Genes for Expression Normalization in Echinococcus spp. Larval Stages  [PDF]
Sergio Martin Espínola, Henrique Bunselmeyer Ferreira, Arnaldo Zaha
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0102228
Abstract: In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus.
Validation of Reference Genes for mRNA Quantification in Adjuvant Arthritis  [PDF]
Muhammad Ayaz Alam Qureshi, Aisha Siddiqah Ahmed, Jian Li, André Stark, Per Eriksson, Mahmood Ahmed
Open Journal of Rheumatology and Autoimmune Diseases (OJRA) , 2012, DOI: 10.4236/ojra.2012.23013
Abstract: Real time quantitative PCR (RT-qPCR) requires a method to normalize the expression of target genes against an en- dogenous reference gene. It is known that commonly used housekeeping genes (HKGs) vary tremendously in inflam- matory conditions; however information about the stability and expression of HKGs in chronic inflammatory joint dis- ease such as rheumatoid arthritis (RA) is scarce. The expressional stability of 10 commonly used HKGs was analyzed in the neuronal (spinal cord, dorsal root ganglia) and in the musculoskeletal tissues (tendon, muscle, epiphysis, capsule, periosteum and ankle joint) using RT-qPCR in the rat model of RA. In individual tissues, suitable HKGs were selected by | △Ct| (│Ct control-Ct arthritis│) and further analyzed by using software programs; geNorm and normfinder. We found hypoxanthine-guanine phosphoribosyl tranferase (HPRT) as the most stable gene except ankle joint while glyce-raldehyde-3-phosphate dehydrogenase (GAPDH) was found as the least stable gene in musculoskeletal tissues. In in-flamed ankle joint where no reference gene was found to be stably expressed, an inflammatory cell marker CD3 was used to normalize peptidylprolyl isomerase B (PPIB), the most homogenous HKG identified among the 10 HKGs. The normalized PPIB was then used to analyze the gene expression of neurokinin 1 (NK1), receptor of substance P, a potent pro-inflammatory mediator. We observed a 3.5 fold increase (p = 0.009) in NK1 expression in inflamed ankle joint compared to control. Our results indicate that reference genes stability should be evaluated before using them as refer- ence during inflammatory conditions. In tissues with intense inflammatory cell infiltration, an inflammatory cell marker should be used to normalize the selected reference gene to avoid erroneous results.
Validation of a set of reference genes to study response to herbicide stress in grasses
Cécile Petit, Fanny Pernin, Jean-Marie Heydel, Christophe Délye
BMC Research Notes , 2012, DOI: 10.1186/1756-0500-5-18
Abstract: The stability of 11 candidate reference genes was assessed in plants resistant or sensitive to herbicides subjected or not to herbicide stress using the complementary statistical methods implemented by NormFinder, BestKeeper and geNorm. Ubiquitin, beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase were identified as the best reference genes. The reference gene set accuracy was confirmed by analysing the expression of the gene encoding acetyl-coenzyme A carboxylase, a major herbicide target enzyme, and of an herbicide-induced gene encoding a glutathione-S-transferase.This is the first study describing a set of reference genes (ubiquitin, beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase) with a stable expression under herbicide stress in grasses. These genes are also candidate reference genes of choice for studies seeking to identify stress-responsive genes in grasses.Differences in gene expression are at the root of plant adaptive response to the environment. Analysing gene expression patterns requires tools enabling sensitive, precise, and reproducible quantification of specific mRNAs. Quantitative real-time polymerase chain reaction (qPCR) is currently the technique of choice for this purpose [1]. However, technical and sample variations usually render absolute quantification of gene expression unreliable. Thus, a normalisation strategy using one, or preferably several, reference gene(s) is generally implemented in qPCR studies [1-4]. A reference gene must be constitutively and constantly expressed in all experimental conditions and samples studied [5,6]. Compared to animals, relatively few studies so far had described validated sets of reference genes in plants [7], and most of them considered species with sequenced genomes (i.e., crop or model species). Exceptions are the grass species Lolium sp. (e.g. [8,9]) and Brachiaria brizantha [10].Alopecurus myosuroides (black-grass, Poaceae) is a grass weed of major economic importance in winter crops in
Selection and Validation of Reference Genes for miRNA Expression Studies during Porcine Pregnancy  [PDF]
Jocelyn M. Wessels, Andrew K. Edwards, Candace Zettler, Chandrakant Tayade
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0028940
Abstract: MicroRNAs comprise a family of small non-coding RNAs that modulate several developmental and physiological processes including pregnancy. Their ubiquitous presence is confirmed in mammals, worms, flies and plants. Although rapid advances have been made in microRNA research, information on stable reference genes for validation of microRNA expression is still lacking. Real time PCR is a widely used tool to quantify gene transcripts. An appropriate reference gene must be chosen to minimize experimental error in this system. A small difference in miRNA levels between experimental samples can be biologically meaningful as these entities can affect multiple targets in a pathway. This study examined the suitability of six commercially available reference genes (RNU1A, RNU5A, RNU6B, SNORD25, SCARNA17, and SNORA73A) in maternal-fetal tissues from healthy and spontaneously arresting/dying conceptuses from sows were separately analyzed at gestation day 20. Comparisons were also made with non-pregnant endometrial tissues from sows. Spontaneous fetal loss is a prime concern to the commercial pork industry. Our laboratory has previously identified deficits in vasculature development at maternal-fetal interface as one of the major participating causes of fetal loss. Using this well-established model, we have extended our studies to identify suitable microRNA reference genes. A methodical approach to assessing suitability was adopted using standard curve and melting curve analysis, PCR product sequencing, real time PCR expression in a panel of gestational tissues, and geNorm and NormFinder analysis. Our quantitative real time PCR analysis confirmed expression of all 6 reference genes in maternal and fetal tissues. All genes were uniformly expressed in tissues from healthy and spontaneously arresting conceptus attachment sites. Comparisons between tissue types (maternal/fetal/non-pregnant) revealed significant differences for RNU5A, RNU6B, SCARNA17, and SNORA73A expression. Based on our methodical assessment of all 6 reference genes, results suggest that RNU1A is the most stable reference gene for porcine pregnancy studies.
Selection and validation of endogenous reference genes using a high throughput approach
Ping Jin, Yingdong Zhao, Yvonne Ngalame, Monica C Panelli, Dirk Nagorsen, Vladia Monsurró, Kina Smith, Nan Hu, Hua Su, Phil R Taylor, Francesco M Marincola, Ena Wang
BMC Genomics , 2004, DOI: 10.1186/1471-2164-5-55
Abstract: Several genes were identified whose expression was highly stable across all samples studied. The usefulness of 8 genes among them was tested by normalizing the relative gene expression against test genes whose expression pattern was known. The range of accuracy of individual endogenous reference genes was wide whereas consistent information could be obtained when information pooled from different endogenous reference genes was used.This study suggests that even when the most stably expressed genes in array experiments are used as endogenous reference, significant variation in test gene expression estimates may occur and the best normalization is achieved when data from several endogenous reference genes are pooled together to minimize minimal but significant variation among samples. We are presently optimizing strategies for the preparation of endogenous reference gene mixtures that could yield information comparable to that of data pooled from individual endogenous reference gene normalizations.Endogenous reference alse referred to as house keeping genes defines in biology the theoretical assumption that certain genes are ubiquitously expressed in nucleated cells possibly because their stable expression is essential for cell survival and welfare in all physio-pathological circumstances. In practical terms, endogenous reference genes provide a useful constant reference to normalize the expression of test genes in different tissues and in different conditions. This is obviously important when estimates of gene expression are provided in relative terms rather than absolute units of measurement. Thus, endogenous reference genes are used as common denominator in biological fractions where the expression of a test gene is described as the relative ratio over an arbitrarily selected internal control presumed to be stably expressed in all circumstances relevant to the experiment [1-3]. Most frequently, glyceraldehydes-3-phosphate dehydrogenase (GAPDH) [4,5], albumin (for h
Validation of Reference Genes in Solenopsis invicta in Different Developmental Stages, Castes and Tissues  [PDF]
Daifeng Cheng, Zhiling Zhang, Xiaofang He, Guangwen Liang
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0057718
Abstract: To accurately assess gene expression levels, it is essential to normalize real-time quantitative PCR (RT-qPCR) data with suitable internal reference genes. For the red imported fire ant, Solenopsis invicta, reliable reference genes to assess the transcript expression levels of the target genes have not been previously investigated. In this study, we examined the expression levels of five candidate reference genes (rpl18, ef1-beta, act, GAPDH, and tbp) in different developmental stages, castes and tissues of S. invicta. To evaluate the suitability of these genes as endogenous controls, three software-based approaches (geNorm, BestKeeper and NormFinder) and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. Furthermore, the optimal number of reference gene(s) was determined by the pairwise variation value. Our data showed that two of the five candidate genes, rpl18 and ef1-beta, were the most suitable reference genes because they have the most stable expression among different developmental stages, castes and tissues in S. invicta. Although widely used as reference gene in other species, in S. invicta the act gene has high variation in expression and was consequently excluded as a reliable reference gene. The two validated reference genes, rpl18 and ef1-beta, can be widely used for quantification of target gene expression with RT-qPCR technology in S. invicta.
Identification and validation of reference genes for quantitative RT-PCR normalization in wheat
Anna R Paolacci, Oronzo A Tanzarella, Enrico Porceddu, Mario Ciaffi
BMC Molecular Biology , 2009, DOI: 10.1186/1471-2199-10-11
Abstract: The expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from 24 different plant samples, which included different tissues, developmental stages and temperature stresses. The selected sequences included 12 well-known HKGs representing different functional classes and 20 genes novel with reference to the normalization issue. The expression stability of the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five different data-sets. Some discrepancies were detected in the ranking of the candidate reference genes, but there was substantial agreement between the groups of genes with the most and least stable expression. Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of suitable normalization genes and can be negatively affected by the use of traditional HKGs with unstable expression, such as actin and α-tubulin.The present research represents the first wide screening aimed to the identification of reference genes and of the corresponding primer pairs specifically designed for gene expression studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes outperformed the traditional HKGs in terms of expression stability under all the tested conditions. The new reference genes will enable more accurate normalization and quantification of gene expression in wheat and will be helpful for designing primer pairs targeting orthologous genes in other plant species.Transcriptome and gene expression analyses are contributing to substantially improve our understanding of the signalling and metabolic pathways underlying developmental and cellular processes. Quantitative RT-PCR (qRT-PCR) is currently one of the most powerful and sensitive techniques for analyzing gene expr
“Datamining” dos genes da celulose sintase relacionados com ESTs de Eucalyptus spp. (Nota Científica). Cellulose synthase genes dataming related with Eucalyptus spp. expressed sequence tags. (SCIENTIFIC NOTE)  [PDF]
Léo ZIMBACK,Edson Seizo MORI,Mário Luiz Teixeira de MORAES,Edson Luiz FURTADO
Revista do Instituto Florestal , 2008,
Abstract: Trata-se de um estudo sobre “datamining”envolvendo genes ligados ao crescimento decontrole n o hormonal, utilizando o banco dedados de ESTs de eucalipto, efetuado atravésdo Projeto Genoma do Eucalipto (FORESTs)comparados ao nível de aminoácidos. Foramidentificados os clusters de ESTsEGBGFB1211D01.g, EGEZRT6201E10.g,EGCCFB1220G07.g, EGRFCL1206E01.g,EGEQST2006A06.g, EGRFCL1206E01.g,EGEQRT3001H05.b e EGBFRT3106G11.g,similares às proteínas de celulose sintase e suassubunidades controlando o crescimento emArabidopsis thaliana, Gossipium hirsutum,Populus tremuloides, Zea mays e Nicotiana alata,registradas no National Center of BiotechnologiesInformation - NCBI, informa o valiosa parafuturos programas de melhoramento genético dogênero Eucalyptus.This is a study about data mining ofexpressed sequence tags (ESTs) involved withcellulose synthase growth effect genes resultedfrom the Eucalyptus ESTs Genome Project(FORESTs) compared at aminoacids level. Using asequencing of derived from cDNAs librariesinduced and not induced by bacteria, wereidentified EST clusters EGBGFB1211D01.g,EGEZRT6201E10.g, EGCCFB1220G07.g,EGRFCL1206E01.g, EGEQST2006A06.g,EGRFCL1206E01.g, EGEQRT3001H05.b, andEGBFRT3106G11.g, similar to cellulose synthaseproteins controlling growth effect in Arabidopsisthaliana, Gossipium hirsutum, Populustremuloides, Zea mays, and Nicotiana alata,registered on National Center of BiotechnologiesInformation - NCBI. These mining results areimportant to improve Eucalyptus breeding programs.
Identification and Validation of Reference Genes for Transcript Normalization in Strawberry (Fragaria × ananassa) Defense Responses  [PDF]
Francisco Amil-Ruiz, José Garrido-Gala, Rosario Blanco-Portales, Kevin M. Folta, Juan Mu?oz-Blanco, José L. Caballero
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0070603
Abstract: Strawberry (Fragaria spp) is an emerging model for the development of basic genomics and recombinant DNA studies among rosaceous crops. Functional genomic and molecular studies involve relative quantification of gene expression under experimental conditions of interest. Accuracy and reliability are dependent upon the choice of an optimal reference control transcript. There is no information available on validated endogenous reference genes for use in studies testing strawberry-pathogen interactions. Thirteen potential pre-selected strawberry reference genes were tested against different tissues, strawberry cultivars, biotic stresses, ripening and senescent conditions, and SA/JA treatments. Evaluation of reference candidate’s suitability was analyzed by five different methodologies, and information was merged to identify best reference transcripts. A combination of all five methods was used for selective classification of reference genes. The resulting superior reference genes, FaRIB413, FaACTIN, FaEF1α and FaGAPDH2 are strongly recommended as control genes for relative quantification of gene expression in strawberry. This report constitutes the first systematic study to identify and validate optimal reference genes for accurate normalization of gene expression in strawberry plant defense response studies.
Selection and Validation of Reference Genes for Gene Expression Analysis in Switchgrass (Panicum virgatum) Using Quantitative Real-Time RT-PCR  [PDF]
Jacinta Gimeno, Nicholas Eattock, Allen Van Deynze, Eduardo Blumwald
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0091474
Abstract: Switchgrass (Panicum virgatum) has received a lot of attention as a forage and bioenergy crop during the past few years. Gene expression studies are in progress to improve new traits and develop new cultivars. Quantitative real time PCR (qRT-PCR) has emerged as an important technique to study gene expression analysis. For accurate and reliable results, normalization of data with reference genes is essential. In this work, we evaluate the stability of expression of genes to use as reference for qRT-PCR in the grass P. virgatum. Eleven candidate reference genes, including eEF-1α, UBQ6, ACT12, TUB6, eIF-4a, GAPDH, SAMDC, TUA6, CYP5, U2AF, and FTSH4, were validated for qRT-PCR normalization in different plant tissues and under different stress conditions. The expression stability of these genes was verified by the use of two distinct algorithms, geNorm and NormFinder. Differences were observed after comparison of the ranking of the candidate reference genes identified by both programs but eEF-1α, eIF-4a, CYP5 and U2AF are ranked as the most stable genes in the samples sets under study. Both programs discard the use of SAMDC and TUA6 for normalization. Validation of the reference genes proposed by geNorm and NormFinder were performed by normalization of transcript abundance of a group of target genes in different samples. Results show similar expression patterns when the best reference genes selected by both programs were used but differences were detected in the transcript abundance of the target genes. Based on the above research, we recommend the use of different statistical algorithms to identify the best reference genes for expression data normalization. The best genes selected in this study will help to improve the quality of gene expression data in a wide variety of samples in switchgrass.
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