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Rajeev K Varshney - One of the best experts on this subject based on the ideXlab platform.
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selection and validation of Housekeeping genes as reference for gene expression studies in pigeonpea cajanus cajan under heat and salt stress conditions
Frontiers in Plant Science, 2015Co-Authors: Pallavi Sinha, Rachit K Saxena, Vikas Singh, L Krishnamurthy, Rajeev K VarshneyAbstract:To identify stable Housekeeping genes as a reference for expression analysis under heat and salt stress conditions in pigeonpea, the relative expression variation for 10 commonly used Housekeeping genes (EF1α, UBQ10, GAPDH, 18Sr RNA, 25Sr RNA, TUB6, ACT1, IF4α, UBC, and HSP90) was studied in root, stem, and leaves tissues of Asha (ICPL 87119), a leading pigeonpea variety. Three statistical algorithms geNorm, NormFinder, and BestKeeper were used to define the stability of candidate genes. Under heat stress, UBC, HSP90, and GAPDH were found to be the most stable reference genes. In the case of salinity stress, GAPDH followed by UBC and HSP90 were identified to be the most stable reference genes. Subsequently, the above identified genes were validated using qRT-PCR based gene expression analysis of two universal stress-resposive genes namely uspA and uspB. The relative quantification of these two genes varied according to the internal controls (most stable, least stable, and combination of most stable and least stable Housekeeping genes) and thus confirmed the choice as well as validation of internal controls in such experiments. The identified and validated Housekeeping genes will facilitate gene expression studies under heat and salt stress conditions in pigeonpea.
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evaluation and validation of Housekeeping genes as reference for gene expression studies in pigeonpea cajanus cajan under drought stress conditions
PLOS ONE, 2015Co-Authors: Pallavi Sinha, Rachit K Saxena, Vikas Singh, L Krishnamurthy, V Suryanarayana, Rajeev K VarshneyAbstract:Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique and its sensitivity depends on the stable performance of reference gene(s) used in the study. A number of Housekeeping genes have been used in various expression studies in many crops however, their expression were found to be inconsistent under different stress conditions. As a result, species specific Housekeeping genes have been recommended for different expression studies in several crop species. However, such specific Housekeeping genes have not been reported in the case of pigeonpea (Cajanus cajan) despite the fact that genome sequence has become available for the crop. To identify the stable Housekeeping genes in pigeonpea for expression analysis under drought stress conditions, the relative expression variations of 10 commonly used Housekeeping genes (EF1α, UBQ10, GAPDH, 18SrRNA, 25SrRNA, TUB6, ACT1, IF4α, UBC and HSP90) were studied on root, stem and leaves tissues of Asha (ICPL 87119). Three statistical algorithms geNorm, NormFinder and BestKeeper were used to define the stability of candidate genes. geNorm analysis identified IF4α and TUB6 as the most stable Housekeeping genes however, NormFinder analysis determined IF4α and HSP90 as the most stable Housekeeping genes under drought stress conditions. Subsequently validation of the identified candidate genes was undertaken in qRT-PCR based gene expression analysis of uspA gene which plays an important role for drought stress conditions in pigeonpea. The relative quantification of the uspA gene varied according to the internal controls (stable and least stable genes), thus highlighting the importance of the choice of as well as validation of internal controls in such experiments. The identified stable and validated Housekeeping genes will facilitate gene expression studies in pigeonpea especially under drought stress conditions.
Gloria V Callard - One of the best experts on this subject based on the ideXlab platform.
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characterization of Housekeeping genes in zebrafish male female differences and effects of tissue type developmental stage and chemical treatment
BMC Molecular Biology, 2008Co-Authors: Amy Mccurley, Gloria V CallardAbstract:Background Research using the zebrafish model has experienced a rapid growth in recent years. Although real-time reverse transcription PCR (QPCR), normalized to an internal reference ("Housekeeping") gene, is a frequently used method for quantifying gene expression changes in zebrafish, many commonly used Housekeeping genes are known to vary with experimental conditions. To identify Housekeeping genes that are stably expressed under different experimental conditions, and thus suitable as normalizers for QPCR in zebrafish, the present study evaluated the expression of eight commonly used Housekeeping genes as a function of stage and hormone/toxicant exposure during development, and by tissue type and sex in adult fish.
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characterization of Housekeeping genes in zebrafish male female differences and effects of tissue type developmental stage and chemical treatment
BMC Molecular Biology, 2008Co-Authors: Amy Mccurley, Gloria V CallardAbstract:Research using the zebrafish model has experienced a rapid growth in recent years. Although real-time reverse transcription PCR (QPCR), normalized to an internal reference ("Housekeeping") gene, is a frequently used method for quantifying gene expression changes in zebrafish, many commonly used Housekeeping genes are known to vary with experimental conditions. To identify Housekeeping genes that are stably expressed under different experimental conditions, and thus suitable as normalizers for QPCR in zebrafish, the present study evaluated the expression of eight commonly used Housekeeping genes as a function of stage and hormone/toxicant exposure during development, and by tissue type and sex in adult fish. QPCR analysis was used to quantify mRNA levels of bactin1, tubulin alpha 1(tuba1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), glucose-6-phosphate dehydrogenase (g6pd), TATA-box binding protein (tbp), beta-2-microglobulin (b2m), elongation factor 1 alpha (elfa), and 18s ribosomal RNA (18s) during development (2 – 120 hr postfertilization, hpf); in different tissue types (brain, eye, liver, heart, muscle, gonads) of adult males and females; and after treatment of embryos/larvae (24 – 96 hpf) with commonly used vehicles for administration and agents that represent known environmental endocrine disruptors. All genes were found to have some degree of variability under the conditions tested here. Rank ordering of expression stability using geNorm analysis identified 18s, b2m, and elfa as most stable during development and across tissue types, while gapdh, tuba1, and tpb were the most variable. Following chemical treatment, tuba1, bactin1, and elfa were the most stably expressed whereas tbp, 18s, and b2m were the least stable. Data also revealed sex differences that are gene- and tissue-specific, and treatment effects that are gene-, vehicle- and ligand-specific. When the accuracy of QPCR analysis was tested using different reference genes to measure suppression of cyp19a1b by an estrogen receptor antagonist and induction of cyp1a by an arylhydrocarbon receptor agonist, the direction and magnitude of effects with stable and unstable genes differed. This study provides data that can be expected to aid zebrafish researchers in their initial choice of Housekeeping genes for future studies, but underlines the importance of further validating Housekeeping genes for each new experimental paradigm and fish species.
Gert Simonet - One of the best experts on this subject based on the ideXlab platform.
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identification and validation of Housekeeping genes in brains of the desert locust schistocerca gregaria under different developmental conditions
BMC Molecular Biology, 2009Co-Authors: Matthias B Van Hiel, Jozef Vanden Broeck, Sofie Van Soest, Pieter Van Wielendaele, Roger Huybrechts, Liesbet Temmerman, Kristel Vuerinckx, Gert SimonetAbstract:To obtain reliable quantitative RT-PCR data, normalization relative to stable Housekeeping genes is required. However, in practice, expression levels of 'typical' Housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in insects are extremely rare and have never been performed in locusts. In this study, putative Housekeeping genes were identified in the desert locust, Schistocerca gregaria and two different software programs (geNorm and Normfinder) were applied to assess the stability of thesegenes. We have identified seven orthologs of commonly used Housekeeping genes in the desert locust. The selected genes were the orthologs of actin, EF1a, GAPDH, RP49, TubA1, Ubi, and CG13220. By employing real time RT-PCR we have analysed the expression of these Housekeeping genes in brain tissue of fifth instar nymphs and adults. In the brain of fifth instar nymphs geNorm indicated Sg-EF1a, Sg-GAPDH and Sg-RP49 as most stable genes, while Normfinder ranked Sg-RP49, Sg-EF1a and Sg-ACT as most suitable candidates for normalization. The best normalization candidates for gene expression studies in the brains of adult locusts were Sg-EF1a, Sg-GAPDH and Sg-Ubi according to geNorm, while Normfinder determined Sg-GAPDH, Sg-Ubi and Sg-ACT as the most stable Housekeeping genes. To perform transcript profiling studies on brains of the desert locust, the use of Sg-RP49, Sg-EF1a and Sg-ACT as reference genes is proposed for studies of fifth instar nymphs. In experiments with adult brains, however, the most preferred reference genes were Sg-GAPDH, Sg-Ubi and Sg-EF1a. These data will facilitate transcript profiling studies in desert locusts and provide a good starting point for the initial selection of genes for validation studies in other insects.
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identification and validation of Housekeeping genes in brains of the desert locust schistocerca gregaria under different developmental conditions
BMC Molecular Biology, 2009Co-Authors: Matthias B Van Hiel, Jozef Vanden Broeck, Sofie Van Soest, Pieter Van Wielendaele, Roger Huybrechts, Liesbet Temmerman, Kristel Vuerinckx, Gert SimonetAbstract:Background To obtain reliable quantitative RT-PCR data, normalization relative to stable Housekeeping genes is required. However, in practice, expression levels of 'typical' Housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in insects are extremely rare and have never been performed in locusts. In this study, putative Housekeeping genes were identified in the desert locust, Schistocerca gregaria and two different software programs (geNorm and Normfinder) were applied to assess the stability of thesegenes.
Claudio Orlando - One of the best experts on this subject based on the ideXlab platform.
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quantitative real time reverse transcription polymerase chain reaction normalization to rrna or single Housekeeping genes is inappropriate for human tissue biopsies
Analytical Biochemistry, 2002Co-Authors: Carmela Tricarico, Pamela Pinzani, Simonetta Bianchi, Milena Paglierani, V Distante, Mario Pazzagli, Stephen A Bustin, Claudio OrlandoAbstract:Careful normalization is essential when using quantitative reverse transcription polymerase chain reaction assays to compare mRNA levels between biopsies from different individuals or cells undergoing different treatment. Generally this involves the use of internal controls, such as mRNA specified by a Housekeeping gene, ribosomal RNA (rRNA), or accurately quantitated total RNA. The aim of this study was to compare these methods and determine which one can provide the most accurate and biologically relevant quantitative results. Our results show significant variation in the expression levels of 10 commonly used Housekeeping genes and 18SrRNA, both between individuals and between biopsies taken from the same patient. Furthermore, in 23 breast cancers samples mRNA and protein levels of a regulated gene, vascular endothelial growth factor (VEGF), correlated only when normalized to total RNA, as did microvessel density. Finally, mRNA levels of VEGF and the most popular Housekeeping gene, glyceraldehyde3-phosphate dehydrogenase (GAPDH), were significantly correlated in the colon. Our results suggest that the use of internal standards comprising single Housekeeping genes or rRNA is inappropriate for studies involving tissue biopsies.
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quantitative real time reverse transcription polymerase chain reaction normalization to rrna or single Housekeeping genes is inappropriate for human tissue biopsies
Analytical Biochemistry, 2002Co-Authors: Carmela Tricarico, Pamela Pinzani, Simonetta Bianchi, Milena Paglierani, V Distante, Mario Pazzagli, Stephen A Bustin, Claudio OrlandoAbstract:Careful normalization is essential when using quantitative reverse transcription polymerase chain reaction assays to compare mRNA levels between biopsies from different individuals or cells undergoing different treatment. Generally this involves the use of internal controls, such as mRNA specified by a Housekeeping gene, ribosomal RNA (rRNA), or accurately quantitated total RNA. The aim of this study was to compare these methods and determine which one can provide the most accurate and biologically relevant quantitative results. Our results show significant variation in the expression levels of 10 commonly used Housekeeping genes and 18S rRNA, both between individuals and between biopsies taken from the same patient. Furthermore, in 23 breast cancers samples mRNA and protein levels of a regulated gene, vascular endothelial growth factor (VEGF), correlated only when normalized to total RNA, as did microvessel density. Finally, mRNA levels of VEGF and the most popular Housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were significantly correlated in the colon. Our results suggest that the use of internal standards comprising single Housekeeping genes or rRNA is inappropriate for studies involving tissue biopsies.
Paul N Span - One of the best experts on this subject based on the ideXlab platform.
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normalization of gene expression measurements in tumor tissues comparison of 13 endogenous control genes
Laboratory Investigation, 2005Co-Authors: Jacques B De Kok, Rian Roelofs, Belinda A J Giesendorf, Jeroen L Pennings, E T Waas, Ton Feuth, Dorine W Swinkels, Paul N SpanAbstract:For interpretation of quantitative gene expression measurements in clinical tumor samples, a normalizer is necessary to correct expression data for differences in cellular input, RNA quality, and RT efficiency between samples. In many studies, a single Housekeeping gene is used for normalization. However, no unequivocal single reference gene (with proven invariable expression between cells) has been identified yet. As the best alternative, the mean expression of multiple Housekeeping genes can be used for normalization. In this study, no attempt was made to determine the gold-standard gene for normalization, but to identify the best single Housekeeping gene that could accurately replace the measurement of multiple genes. Expression patterns of 13 frequently used Housekeeping genes were determined in 80 normal and tumor samples from colorectal, breast, prostate, skin, and bladder tissues with real-time quantitative RT-PCR. These genes included, large ribosomal protein, beta-actin, cyclophilin A, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerokinase 1, beta-2-microglobin, beta-glucuronidase, hypoxanthine ribosyltransferase (HPRT), TATA-box-binding protein, transferrin receptor, porphobilinogen deaminase, ATP synthase 6, and 18S ribosomal RNA. Principal component analysis was used to analyze these expression patterns, independent of the level of expression. Our approach identified HPRT as the single best reference gene that could be used as an accurate and economic alternative for the measurement of multiple Housekeeping genes. We recommend this gene for future studies to standardize gene expression measurements in cancer research and tumor diagnostics until a definite gold standard has been determined.
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normalization of gene expression measurements in tumor tissues comparison of 13 endogenous control genes
Laboratory Investigation, 2005Co-Authors: Rian Roelofs, Belinda A J Giesendorf, Jeroen L Pennings, E T Waas, Ton Feuth, Dorine W Swinkels, Paul N SpanAbstract:For interpretation of quantitative gene expression measurements in clinical tumor samples, a normalizer is necessary to correct expression data for differences in cellular input, RNA quality, and RT efficiency between samples. In many studies, a single Housekeeping gene is used for normalization. However, no unequivocal single reference gene (with proven invariable expression between cells) has been identified yet. As the best alternative, the mean expression of multiple Housekeeping genes can be used for normalization. In this study, no attempt was made to determine the gold-standard gene for normalization, but to identify the best single Housekeeping gene that could accurately replace the measurement of multiple genes. Expression patterns of 13 frequently used Housekeeping genes were determined in 80 normal and tumor samples from colorectal, breast, prostate, skin, and bladder tissues with real-time quantitative RT-PCR. These genes included, large ribosomal protein, β-actin, cyclophilin A, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerokinase 1, β-2-microglobin, β-glucuronidase, hypoxanthine ribosyltransferase (HPRT), TATA-box-binding protein, transferrin receptor, porphobilinogen deaminase, ATP synthase 6, and 18S ribosomal RNA. Principal component analysis was used to analyze these expression patterns, independent of the level of expression. Our approach identified HPRT as the single best reference gene that could be used as an accurate and economic alternative for the measurement of multiple Housekeeping genes. We recommend this gene for future studies to standardize gene expression measurements in cancer research and tumor diagnostics until a definite gold standard has been determined.