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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which plays an important role in central carbon metabolism, was studied in Aeluropus lagopoides, which is an important perennial grass found in drought-stricken areas and deserts f...
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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which plays an important role in central carbon metabolism, was studied in Aeluropus lagopoides, which is an important perennial grass found in drought-stricken areas and deserts from Africa to South Asia. Aeluropus lagopoides is an ideal candidate plant system to identify novel or enhanced stress tolerance mechanisms. The GAPDH has stable expression under different conditions and it is a typical housekeeping gene in prokaryotes and eukaryotes. So far, there are no reports about identification of housekeeping genes for gene expression analysis in Aeluropus lagopoides. Thus in the present study, a GAPDH cDNA was cloned and sequenced from this plant. Our results revealed that the open reading frame (ORF) (1014 bp) encoded a polypeptide of 337 amino-acid residues. The deduced amino acid sequence indicated a protein with a molecular weight of 364.95 kDa and estimated isoelectric point value of 7.06. Multiple alignments of putative amino acid sequences indicated high similarity between Aeluropus lagopoides GAPDH and analogous protein derived from other plants, particularly monocots. Nucleotide blast and phylogenetic analysis revealed that this plant had maximum identity with Cleistogenes songorica. Results of this experiment can help us do transcriptional analysis by qRT-PCR to identify stress-tolerance mechanisms.
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In this study, to systematically identify the most stably expressed genes for internal reference in zebrafish Danio rerio investigations, 37 D. rerio transcriptomic datasets (both RNA sequencing and microarray data) were collected...
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In this study, to systematically identify the most stably expressed genes for internal reference in zebrafish Danio rerio investigations, 37 D. rerio transcriptomic datasets (both RNA sequencing and microarray data) were collected from gene expression omnibus (GEO) database and unpublished data, and gene expression variations were analysed under three experimental conditions: tissue types, developmental stages and chemical treatments. Forty-four putative candidate genes were identified with the c.v. <0.2 from all datasets. Following clustering into different functional groups, 21 genes, in addition to four conventional housekeeping genes (eef1a1l1, b2m, hrpt1l and actb1), were selected from different functional groups for further quantitative real-time (qrt-)PCR validation using 25 RNA samples from different adult tissues, developmental stages and chemical treatments. The qrt-PCR data were then analysed using the statistical algorithm refFinder for gene expression stability. Several new candidate genes showed better expression stability than the conventional housekeeping genes in all three categories. It was found that sep15 and metap1 were the top two stable genes for tissue types, ube2a and tmem50a the top two for different developmental stages, and rpl13a and rp1p0 the top two for chemical treatments. Thus, based on the extensive transcriptomic analyses and qrt-PCR validation, these new reference genes are recommended for normalization of D. rerio qrt-PCR data respectively for the three different experimental conditions. (C) 2016 The Fisheries Society of the British Isles
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Background DBA/1 mice arthritis models have contributed to our understanding of human rheumatoid arthritis (RA) and spondyloarthritis (SpA) pathogenesis, as well as the exploration of therapeutic targets for treatment. Quantitativ...
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Background DBA/1 mice arthritis models have contributed to our understanding of human rheumatoid arthritis (RA) and spondyloarthritis (SpA) pathogenesis, as well as the exploration of therapeutic targets for treatment. Quantitative polymerase chain reaction (qPCR) is an indispensable tool in molecular research, which requires reference gene validation to obtain consistent and reliable results. Objective To determine the stability of candidate reference genes for qPCR in the joint of collagen-induced arthritis (CIA) and spontaneous arthritis (SpAD) DBA/1 mice. Methods The expression of eleven commonly used reference genes (ACTB, B2M, EF1a, GAPDH, HMBS, HPRT, PPIB, RPL13A, SDHA, TBP, and YWHAZ) was assessed by qPCR and the data were compared using delta-Ct methods and the geNorm, NormFinder, and RefFinder software packages. Genes identified as stable in each model were used for the quantification of inflammatory cytokines Results The gene stabilities differed between the two arthritis models in the DBA/1 mice. EF1a and RPL13A were the best reference genes for SpAD, while RPL13A and TBP were the best for the CIA. These genes allowed the data normalization for the quantification of the inflammatory cytokines in both models; these results showed an increase in the expression of IL-1B, IL-12B, IL-17A, and IL-6 in the inflamed joints. The use of different primer sequences for the same reference gene resulted in different relative quantification values. Conclusion This study demonstrates that commonly used reference genes may not be suitable for arthritic tissues from DBA/1 mice, and strengthening the principle that meticulous validation of reference genes is essential before each experiment to obtain valid and reproducible qPCR data for analysis or interpretation.
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Bacteria belonging to the genus Bradyrhizobium nodulate various leguminous woody plants and herbs, including economically important crops such as soybean, peanut and cowpea. Here we analysed 39 Bradyrhizobium strains originating f...
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Bacteria belonging to the genus Bradyrhizobium nodulate various leguminous woody plants and herbs, including economically important crops such as soybean, peanut and cowpea. Here we analysed 39 Bradyrhizobium strains originating from root nodules of the leguminous trees and crops Acacia saligna, Faidherbia albida, Erythrina brucei, Albizia gummifera, Millettia ferruginea, Cajanus cajan, Vigna unguiculata and Phaseolus vulgaris, growing in southern Ethiopia. Multilocus sequence analyses (MLSA) of the 16S rRNA, gird), recA,gyrB and dnaKgenes and the ITS region grouped the test strains into seven well-supported genospecies (I-VII), six of which occupied distinct positions excluding all hitherto defined Bradyrhizobium species. Analyses of the nodA, nodC and nifH genes suggested different evolutionary history of the chromosomal and symbiosis-related genes. Our study corroborates earlier findings that Ethiopia is a hotspot for rhizobial biodiversity, justifying further search for novel strains from this region and calling for intensified research on the ecology and biochemistry of these organisms. (C ) 2017 Elsevier GmbH. All rights reserved.
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Human housekeeping genes are often confused with essential human genes, and several studies regard both types of genes as having the same level of evolutionary conservation. However, this is not necessarily the case. To clarify th...
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Human housekeeping genes are often confused with essential human genes, and several studies regard both types of genes as having the same level of evolutionary conservation. However, this is not necessarily the case. To clarify this, we compared the differences between human housekeeping genes and essential human genes with respect to four aspects: the evolutionary rate (dN/dS), protein sequence identity, single-nucleotide polymorphism (SNP) density and level of linkage disequilibrium (LD). The results showed that housekeeping genes had lower evolutionary rates, higher sequence identities, lower SNP densities and higher levels of LD compared with essential genes. Together, these findings indicate that housekeeping and essential genes are two distinct types of genes, and that housekeeping genes have a higher level of evolutionary conservation. Therefore, we suggest that researchers should pay careful attention to the distinctions between housekeeping genes and essential genes. Moreover, it is still controversial whether we should substitute human orthologs of mouse essential genes for human essential genes. Therefore, we compared the evolutionary features between human orthologs of mouse essential genes and human housekeeping genes and we got inconsistent results in long-term and short-term evolutionary characteristics implying the irrationality of simply replacing human essential genes with human orthologs of mouse essential genes.
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The family Rhizobiaceae accommodates the seven genera Rhizobium, Neorhizobium, Allorhizobium, Agrobacterium, Ensifer (syn. Sinorhizobium), Shinella and Ciceribacter. However, several so-called Rhizobium species do not exhibit robu...
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The family Rhizobiaceae accommodates the seven genera Rhizobium, Neorhizobium, Allorhizobium, Agrobacterium, Ensifer (syn. Sinorhizobium), Shinella and Ciceribacter. However, several so-called Rhizobium species do not exhibit robust phylogenetic positions. Rhizobium is extremely heterogeneous and is in need of major revision. Therefore, a phylogenetic examination of the family Rhizobiaceae by multilocus sequence analysis (MLSA) of four housekeeping genes among 100 strains of the family was undertaken. Based on the results we propose the delineation of the new genus Pararhizobium in the Rhizobiaceae family, and 13 new species combinations: Agrobacterium nepotum comb. nov., Agrobacterium pusense comb. nov., Agrobacterium skierniewicense comb. nov., Allorhizobium yids comb. nov., Allorhizobium taibaishanense comb. nov., Allorhizobium paknamense comb. nov., Allorhizobium oryzae comb. nov., Allorhizobium pseudoryzae comb. nov., Allorhizobium borbori comb. nov., Pararhizobium giardinii comb. nov., Pararhizobium capsulatum comb. nov., Pararhizobium herbae comb. nov., and Pararhizobium sphaerophysae comb. nov. (C) 2015 Elsevier GmbH. All rights reserved.
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Objective: To investigate the stability of various housekeeping genes (HKG) within healthy versus scarred peritoneal mesothelium. The use of HKG as internal controls for quantitative real-time polymerase chain reaction (qRT-PCR) s...
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Objective: To investigate the stability of various housekeeping genes (HKG) within healthy versus scarred peritoneal mesothelium. The use of HKG as internal controls for quantitative real-time polymerase chain reaction (qRT-PCR) studies is based on the assumption of their inherent stability. However, recent evidence suggests that this is not true for all HKG and that stability may be tissue specific and affected by certain pathologies. Design: Paired mesothelial (n = 10) and adhesion tissue samples (n = 10) were taken during laparoscopic surgery. The stability of 12 candidate reference genes in the mesothelial tissues were evaluated; these include ATP5b, SDHA, CYC1, 18S rRNA, RPL13A, ACTB, YWHAZ, TOP1, UBC, EIF4A2, GAPDH, and B2M. Setting: Hospital. Patient(s): Female patients undergoing laparoscopic gynecological surgery were recruited from the Princess Anne Hospital, United Kingdom. Intervention(s): Assessment of HKG expression stability using geNorm algorithm software. Main Outcome Measure(s): Stability measure (M) generated by geometric averaging of multiple target genes and mean pairwise variation of genes. Result(s): The most stable HKGs observed across both healthy and adhesion-related mesothelium were found to be ACTB, YWHAZ, and CYC1. ACTB had a higher expression in healthy mesothelium compared with in peritoneal adhesion tissue. Conclusion(s): This study indicates that ACTB, YWHAZ, and CYC1 are the appropriate internal controls for qRT-PCR analysis in mesothelial gene expression studies. Published discrepancies in gene expression studies using the mesothelium may therefore be due in part to inappropriate HKG selection.
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This paper aims to discuss various aspects of the use of reference genes in qPCR technique used in the thousands of present studies. Most frequently, these are housekeeping genes and they must meet several criteria so that they ca...
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This paper aims to discuss various aspects of the use of reference genes in qPCR technique used in the thousands of present studies. Most frequently, these are housekeeping genes and they must meet several criteria so that they can lay claim to the name. Lots of papers report that in different conditions, for different organisms and even tissues the basic assumption-the constant level of the expression is not maintained for many genes that seem to be perfect candidates. Moreover, their transcription can not be affected by experimental factors. Sounds simple and clear but a great number of designed protocols and lack of consistency among them brings confusion on how to perform experiment properly. Since during selection of the most stable normalizing gene we can not use any reference gene, different ways and algorithms for their selection were developed. Such methods, including examples of best normalizing genes in some specific cases and possible mistakes are presented based on available sources. Numerous examples of reference genes applications, which are usually in too few numbers in relevant articles not allowing to make a solid fundament for a reader, will be shown along with instructive compilations to make an evidence for presented statements and an arrangement of future qPCR experiments. To include all the pitfalls and problems associated with the normalization methods there is no way not to begin from sample preparation and its storage going through candidate gene selection, primer design and statistical analysis. This is important because numerous short reviews available cover the topic only in lesser extent at the same time giving the reader false conviction of complete topic recognition.
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The aim of this study was to determine the steady-state expression of 13 selected housekeeping genes in the myometrium of cyclic and pregnant cows. Cells taken from bovine myometrium on days 1-5, 6-10, 11-16 and 17-20 of the oestr...
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The aim of this study was to determine the steady-state expression of 13 selected housekeeping genes in the myometrium of cyclic and pregnant cows. Cells taken from bovine myometrium on days 1-5, 6-10, 11-16 and 17-20 of the oestrous cycle and in weeks 3-5, 6-8 and 9-12 of pregnancy were used. Reverse transcribed RNA was amplified in real-time PCR using designed primers. Reaction efficiency was determined with the Linreg programme. The geNorm and NormFinder programmes were used to select the best housekeeping genes. They calculate the expression stability factor for each used housekeeping gene with the smallest value for most stably expressed genes. According to geNorm, the most stable housekeeping genes in the myometrium were C2orf29, TPB and TUBB2B, while the least stably expressed genes were 18S RNA, HPRT1 and GAPDH. NormFinder identified the best genes in the myometrium as C2orf29, MRPL12 and TBP, while the worst genes were 18S RNA, B2M and SF3A1. Differences in stability factors between the two programmes may also indicate that the physiological status of the female, e. g. pregnancy, affects the stability of expression of housekeeping genes. The different expression stability of housekeeping genes did not affect progesterone receptor expression but it could be important if small differences in gene expression were measured between studies.
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Western blot analysis is used for evaluation of the level of proteins production in organs and tissues, and housekeeping proteins GAPDH, actin, and tubulin are usually used as the reference proteins. The signal of the target prote...
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Western blot analysis is used for evaluation of the level of proteins production in organs and tissues, and housekeeping proteins GAPDH, actin, and tubulin are usually used as the reference proteins. The signal of the target protein is normalized to the corresponding signal of the reference protein. The data on the intensity of actin, tubulin, and GAPDH synthesis are fragmentary: their expression differs in different organs and can vary depending on age, which is often not taken into account in experimental studies. We studied the features of the production of reference proteins in the liver, heart, brain, and lungs of newborn, prepubertal, and adult male Wistar rats. Age-related differences in the expression of beta-actin, beta-tubulin, and GAPDH in the myocardium and dorsal prefrontal cortex were revealed. GAPDH expression in the dorsal prefrontal cortex in adult rats was significantly higher than in prepubertal rats; GAPDH expression in the myocardium of adult rats was significantly higher than in newborns. The level of actin in the dorsal prefrontal cortex in newborn rats was significantly higher than in prepubertal and adult rats. In the liver and lungs, the expression of actin, tubulin, and GAPDH did not differ in newborn, prepubertal, and adult rats. When choosing the reference protein for Western blotting, animals age and the studied organ should be taken into account.
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