Evaluation of reference genes at different developmental stages for quantitative real-time PCR in Aedes aegypti
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Abstract The mosquito Aedes aegypti (Ae. aegypti ) is the most notorious vector of illness-causing viruses such as Dengue, Chikugunya, and Zika. Although numerous genetic expression studies utilizing quantitative real-time PCR (qPCR) have been conducted with regards to Ae. aegypti , a panel of genes to be used suitably as references for the purpose of expression-level normalization within this epidemiologically important insect is presently lacking. Here, the usability of seven widely-utilized reference genes i.e. actin ( ACT ), eukaryotic elongation factor 1 alpha ( eEF1α ), alpha tubulin ( α - tubulin ), ribosomal proteins L8, L32 and S17 ( RPL8, RPL32 and RPS17 ), and glyceraldeyde 3-phosphate dehydrogenase ( GAPDH ) were investigated. Expression patterns of the reference genes were observed in sixteen pre-determined developmental stages and in cell culture. Gene stability was inferred from qPCR data through three freely available algorithms i.e. BestKeeper, geNorm, and NormFinder. The consensus rankings generated from stability values provided by these programs suggest a combination of at least two genes for normalization. ACT and RPS17 are the most dependably expressed reference genes and therefore, we propose an ACT/RPS17 combination for normalization in all Ae. aegypti derived samples. GAPDH performed least desirably, and is thus not a recommended reference gene. This study emphasizes the importance of validating reference genes in Ae. aegypti for qPCR based research.Keywords:
Reference Genes
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Glyceraldehyde 3-phosphate dehydrogenase
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Housekeeping gene
Glyceraldehyde 3-phosphate dehydrogenase
Ribosomal protein
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Safflower (Carthamus tinctorius L., Asteraceae) is an important oil crop and medicinal plant. Gene expression analysis is gaining importance in the research of safflower. Quantitative PCR has become a powerful method for gene study. Reference genes are one of the major qualification requirements of qPCR because they can reduce the variability. To identify the reference genes in safflower, nine candidate genes of the housekeeping genes were selected from the EST library of safflower constructed by our lab: CtACT (actin), CtGAPDH (glyceraldehyde 3-phosphate dehydrogenase), CtE1F4A (elongation factor 1 alpha), CtTUA (alpha-tubulin), CtTUB (beta-tubulin), CtPP2A (serine/threonine-protein phosphatase), CtE1F4A (eukaryotic initiation factor 4A), CtUBI (Ubiquitin), and Ct60S (60S acidic ribosomal protein). Expression stability was examined by qPCR across 54 samples, representing tissues at different flowering stages and two chemotype of safflower lines. We assessed the expression stability of these candidate genes by employing four different algorithms (geNorm, NormFinder, ΔCt approach, and BestKeeper) and found that CtUBI and Ct60S were the highly ranked candidate genes. CtUBI and Ct60S were used as reference genes to evaluate the expression of CtFAD2-10 and CtKASII. Our data suggest CtUBI and Ct60S could be used as internal controls to normalize gene expression in safflower.
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Reference genes are commonly used as the endogenous normalisation measure for the relative quantification of target genes. The appropriate application of quantitative real-time PCR (RT-qPCR), however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability. For this purpose, seven reference genes were investigated in tissues of the most important cereals (wheat, barley and oats). Titre of Barley yellow dwarf virus (BYDV) was determined in oats using relative quantification with different reference genes and absolute quantification, and the results were compared. The expression of seven potential reference genes was evaluated in tissues of 180 healthy, physiologically stressed and virus-infected cereal plants. These genes were tested by RT-qPCR and ranked according to the stability of their expression using three different methods (two-way ANOVA, GeNorm and NormFinder tools). In most cases, the expression of all genes did not depend on abiotic stress conditions or virus infections. All the genes showed significant differences in expression among plant species. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUBB) and 18S ribosomal RNA (18S rRNA) always ranked as the three most stable genes. On the other hand, elongation factor-1 alpha (EF1A), eukaryotic initiation factor 4a (EIF4A), and 28S ribosomal RNA (28S rRNA) for barley and oat samples; and alpha-tubulin (TUBA) for wheat samples were consistently ranked as the less reliable controls. The BYDV titre was determined in two oat varieties by RT-qPCR using three different quantification approaches. There were no significant differences between the absolute and relative quantifications, or between quantification using GAPDH + TUBB + TUBA +18S rRNA and EF1A + EIF4A + 28S rRNA. However, there were discrepancies between the results of individual assays. The geometric average of GAPDH, 18S rRNA and TUBB is suitable for normalisation of BYDV quantification in barley tissues. For wheat and oat samples, a combination of four genes is necessary: GAPDH, 18S rRNA, TUBB and EIF4A for wheat; and GAPDH, 18S rRNA, TUBB and TUBA for oat is recommended.
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Barley yellow dwarf
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Glyceraldehyde 3-phosphate dehydrogenase
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Database normalization
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<p class="1Body">Quantitative Real-time Polymerase Chain Reaction (qPCR) is an important tool for molecular biology and biotechnology research, widely used to determine the expression levels of mRNA. Two main methods to performing qPCR are largely used: The absolute quantification, in which the mRNA levels are determined by using a standard curve and the relative method, which is based on the use of reference genes. Reference genes are widely expressed in cells of animal and plant tissues and their expression pattern are theoretically unchanged within several situations, which makes them an excellent choice to normalize mRNA quantification data in relative qPCR studies. However, several reports are increasingly showing that the use of only one reference gene in relative qPCR studies should be avoided, because in the real world their expression levels can significantly change from tissue to tissue. Several softwares, such as geNorm, BestKeeper and NormFinder, have been developed to perform data normalisation, and these programs may assist in choosing the most stable reference genes. The aim of this review was to describe the current normalisation strategies used in qPCR assay, as well as to establish essential rules to perform reliable mRNA quantification. Finally, this review show some innovations in the advances on qPCR.</p>
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Background Quantitative real-time PCR (qPCR) is a widely used technique for gene expression analysis. Its reliability is highly dependent upon selection of the appropriate reference genes for accurate gene expression normalization. In this study, we investigated the expression stability of 10 commonly used reference genes in a mouse myocardial infarction model. Methods & Results The expression stability of the 10 reference genes (Actb, B2m, Eef1a1, Gapdh, Hprt, Polr2a, Ppia, Rpl13a, Tbp, Tpt1) was analyzed using the geNorm software. Overall, the combination of Hprt, Rpl13a and Tpt1 was the most stable reference gene set in our experiments. Gapdh, Polr2a and Actb consistently showed the highest gene expression variability and the expression levels of Gapdh, Polr2a, Actb, B2m and Eef1a1 were found to be selectively up- or downregulated after myocardial infarction. We normalized the expression of Nppb and Vcam1, using different reference gene strategies and demonstrated that their induction after myocardial infarction was most clearly revealed with the optimal reference gene combination. However, the use of suboptimal reference gene combinations resulted in detrimental effects on gene expression levels and variability with a gradual loss of the expression differences and a significant reduction in statistical power. Conclusions Hprt, Rpl13a and Tpt1 are a set of stably expressed reference genes for accurate gene expression normalization in myocardial infarction studies in mice. We found that Gapdh, Polr2a and Actb display high expression variability in mouse myocardial infarction tissues and that loss of statistical power and increase in sample size are the evident consequences of choosing suboptimal combinations of reference genes. We furthermore caution against the use of Gapdh, Polr2a, Actb, B2m and Eef1a1 for gene expression normalization in myocardial infarction studies because of selective up- or downregulation after myocardial infarction, which could potentially lead to biased study outcomes.
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Glyceraldehyde 3-phosphate dehydrogenase
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Abstract The accuracy of reverse transcription quantitative real-time PCR (RT-qPCR) is strongly depended on the stability of reference gene. Callerya speciosa , as a traditionally Chinese medicine, has a long cultivation history in south China. It is essential to select the suitable reference gene to obtain reliable RT-qPCR results when gene expression changes were evaluated. However, suitable reference genes in C. speciosa have not yet been investigated for accurate gene expression quantification under different experimental conditions. In this study, eight candidate reference genes ( GAPDH , 60S , ACTIN , TUA2 , TUB1 , TIF5 , UBQ , EF2 ) were selected from the transcriptome databases, and their expression stability under six experimental conditions (developmental stages, tissues, MeJA treatment, GA 3 treatment, CPPU and PP 333 treatment) was evaluated using ΔCT , geNorm , NormFinder , BestKeeper , RefFinder programs. The results showed that GAPDH was the optimal reference gene for all different experimental conditions, whereas ACTIN showed the most stability under the hormone treatments in C. speciosa . GAPDH and EF2 were proved to be the most stable genes for developmental stages, while different genes ( GAPDH and TUB1 ) were stable reference genes for tissues. For treatments, ACTIN was identified as the most stable gene under most of hormone treatments. TUBI and ACTIN were at the beginning of the ranking order in PP 333 treatment, while GAPDH and ACTIN were adequate for normalization in MeJA and GA 3 treatments. TUBI and GAPDH were the most stable genes for CPPU treatment, while ACTIN was proved to be the most stable gene for three different treatments (MeJA, GA 3 and PP 333 ). Validation of reference genes was carried out by the target gene CsMYB36 , which further confirmed their reliability. These results provided a theoretical basis for subsequent research on the regulation of functional gene expression in C. speciosa.
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Glyceraldehyde 3-phosphate dehydrogenase
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Since blood cells produce various soluble factors like cytokines or chemokines, gene expression analysis in whole blood could be important to investigate disease pathogenesis. In gene expression analysis with quantitative real-time RT-PCR, accurate determination of relative mRNA transcription levels requires appropriate reference genes. To identify the optimal reference gene in canine whole blood, we compared transcription levels of twelve candidate reference genes in total RNA extracted using the PAXgene system. The stability of the reference gene was evaluated by three different statistical programs, GeNorm, Normfinder and Bestkeeper. The results indicated that SDHA, CG14980 and TBP were the most stably expressed genes, which can be used as optimal reference genes for gene expression analysis in canine whole blood.
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SDHA
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Abstract The accuracy of reverse transcription quantitative real-time PCR (RT-qPCR) is strongly depended on the stability of reference gene. Callerya speciosa , as a traditionally Chinese medicine, has a long cultivation history in south China. It is essential to select the suitable reference gene to obtain reliable RT-qPCR results when gene expression changes were evaluated. However, suitable reference genes in C. speciosa have not yet been investigated for accurate gene expression quantification under different experimental conditions. In this study, eight candidate reference genes ( GAPDH , 60S , ACTIN , TUA2 , TUB1 , TIF5 , UBQ , EF2 ) were selected from the transcriptome databases, and their expression stability under six experimental conditions (developmental stages, tissues, MeJA treatment, GA 3 treatment, CPPU and PP 333 treatment) was evaluated using ΔCT , geNorm , NormFinder , BestKeeper , RefFinder programs. The results showed that GAPDH was the optimal reference gene for all different experimental conditions, whereas ACTIN showed the most stability under the hormone treatments in C. speciosa . GAPDH and EF2 were proved to be the most stable genes for developmental stages, while different genes ( GAPDH and TUB1 ) were stable reference genes for tissues. For treatments, ACTIN was identified as the most stable gene under most of hormone treatments. TUBI and ACTIN were at the beginning of the ranking order in PP 333 treatment, while GAPDH and ACTIN were adequate for normalization in MeJA and GA 3 treatments. TUBI and GAPDH were the most stable genes for CPPU treatment, while ACTIN was proved to be the most stable gene for three different treatments (MeJA, GA 3 and PP 333 ). Validation of reference genes was carried out by the target gene CsMYB36 , which further confirmed their reliability. These results provided a theoretical basis for subsequent research on the regulation of functional gene expression in C. speciosa.
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Glyceraldehyde 3-phosphate dehydrogenase
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The selection of reference genes is essential for gene expression studies when using a real-time quantitative polymerase chain reaction (PCR). Reference gene selection should be performed for each experiment because the gene expression level may be changed in different experimental conditions. In this study, the stability of mRNA expression was determined for seven genes: HPRT1, RPS18, NACA, TBP, TAF4B, RPL32 and OAZ1. The stability of these reference genes was investigated in the skeletal muscle tissue of pig foetuses, piglets and adult pigs using real-time quantitative PCR and SYBR green chemistry. The expression of stability of the used reference genes was calculated using the geNorm application. Different gene expression profiles among the age categories of pigs were found out. RPS18 has been identified as the gene with the most stable expression in the muscle tissue of all pig age categories. HPRT1 and RPL32 were found to have the highest stability in piglets and adult pigs, and in foetuses and adults pigs, respectively. The newly used reference gene, TAF4B, reached the highest expression stability in piglets.
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Muscle tissue
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