Evaluation of candidate RT-qPCR reference genes in the aging African turquoise killifish brain

The present study aimed to identify reference genes for qPCR analysis of T.rubrum growth in culture media which promote adhesion-inducing conditions to the host tissue. We investigated the suitability of six candidate reference genes: β-act, β-tub, ef1-α, gapdh, sdha and rpl2 in reference strain of Trichophyton rubrum in response to different environmental stimuli. The stability of these genes was determined by NormFinder, geNorm and BestKeeper software. Our data obtained from the three algorithms revealed that mRNA expression levels of two candidate reference genes, ef1-α and β-tub, remained the most stable in response to different carbon sources, while different sample sets had their own most stable reference genes, highlighting the importance of the choice of internal controls in qPCR experiments. We then checked the stability of ef1-α and β-tub reference genes expression in different T.rubrum strains, suggesting that these two genes are reliable for normalisation of qPCR. Finally, we validated the suitability of selected reference genes as internal controls for target gene (SUB3) using the 2-ΔΔCt method. The best result indicating an increase of SUB3 transcript of T.rubrum was found when the two the most stable reference (ef1-α and β-tub ) genes were used, as revealed by all three algorithms. We recommend the use of ef1-α and β-tub as reference genes for qPCR analysis of target gene expression in T.rubrum exposed to different carbon sources which promote adhesion-inducing conditions.

Rhizophora apiculata is a halophytic, small mangrove tree distributed along the coastal regions of the tropical and subtropical areas of the world. They are natural genetic reservoirs of salt adaptation genes and offer a unique system to explore adaptive mechanisms under salinity stress. However, there are no reliable studies available on selection and validation of reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) in R. apiculata physiological tissues and in salt stress conditions. The selection of appropriate candidate reference gene for normalization of qRT-PCR data is a crucial step towards relative analysis of gene expression. In the current study, seven genes such as elongation factor 1α (EF1α), Ubiquitin (UBQ), β-tubulin (β-TUB), Actin (ACT), Ribulose1,5-bisphosphate carboxylase/oxygenase (rbcL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 18S rRNA (18S) were selected and analyzed for their expression stability. Physiological tissues such as leaf, root, stem, and flower along with salt stress leaf samples were used for selection of candidate reference genes. The high-quality expression data was obtained from biological replicates and further analyzed using five different programs such as geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder. All algorithms comprehensively ranked EF1α followed by ACT as the most stable candidate reference genes in R. apiculata physiological tissues. Moreover, β-TUB and 18S were ranked as moderately stable candidate reference genes, while GAPDH and rbcL were least stable reference genes. Under salt stress, EF1α was comprehensively recommended top-ranked candidate reference gene followed by ACT and 18S. In order to validate the identified most stable candidate reference genes, EF1α, ACT, 18S and UBQ were used for relative gene expression level of sodium/proton antiporter (NHX) gene under salt stress. The expression level of NHX varied according to the internal control which showed the importance of selection of appropriate reference gene. Taken together, this is the first ever systematic attempt of selection and validation of reference gene for qRT-PCR in R. apiculata physiological tissues and in salt stress. This study would promote gene expression profiling of salt stress tolerance related genes in R. apiculata.

Centipedegrass (Eremochloa ophiuroides (Munro.) Hack.) is a species originating in China and is an excellent warm-season turfgrass. As a native species in southern China, it is naturally distributed in the phosphorus-deficient and aluminum-toxic acid soil areas. It is important to research the molecular mechanism of centipedegrass responses to phosphorus-deficiency and/or aluminum-toxicity stress. Quantitative Real-Time PCR (qRT-PCR) is a common method for gene expression analysis, and the accuracy of qRT-PCR results depends heavily on the stability of internal reference genes. However, there are still no reported stable and effective reference genes for qRT-PCR analysis of target genes under the acid-soil-related stresses in different organs of centipedegrass. For scientific rigor, the gene used as a reference for any plant species and/or any stress conditions should be first systematically screened and evaluated. This study is the first to provide a group of reliable reference genes to quantify the expression levels of functional genes of Eremochloa ophiuroides under multiple stresses of P deficiency and/or aluminum toxicity. In this study, centipedegrass seedlings of the acid-soil-resistant strain ‘E041’ and acid-soil-sensitive strain ‘E089’ were used for qRT-PCR analysis. A total of 11 candidate reference genes (ACT, TUB, GAPDH, TIP41, CACS, HNR, EP, EF1α, EIF4α, PP2A and actin) were detected by qRT-PCR technology, and the stability of candidate genes was evaluated with the combination of four internal stability analysis software programs. The candidate reference genes exhibited differential stability of expression in roots, stems and leaves under phosphorus-deficiency and/or aluminum-toxicity stress. On the whole, the results showed that GAPDH, TIP41 and HNR were the most stable in the total of samples. In addition, for different tissues under various stresses, the selected reference genes were also different. CACS and PP2A were identified as two stable reference genes in roots through all three stress treatments (phosphate deficiency, aluminum toxicity, and the multiple stress treatment of aluminum toxicity and phosphate deficiency). Moreover, CACS was also stable as a reference gene in roots under each treatment (phosphate deficiency, aluminum toxicity, or multiple stresses of aluminum toxicity and phosphate deficiency). In stems under all three stress treatments, GAPDH and EIF4α were the most stable reference genes; for leaves, PP2A and TIP41 showed the two highest rankings in all three stress treatments. Finally, qRT-PCR analysis of the expression patterns of the target gene ALMT1 was performed to verify the selected reference genes. The application of the reference genes identified as internal controls for qRT-PCR analysis will enable accurate analysis of the target gene expression levels and expression patterns in centipedegrass under acid-soil-related stresses.

Accurate reflection of hepatopancreas antioxidation and detoxification in Procambarus clarkii during virus infection and drug treatment: Reference gene selection, evaluation and expression analysis

BackgroundAppropriate reference genes are critical to accurately quantifying relative gene expression in research and clinical applications. Numerous efforts have been made to select the most stable reference gene(s), but a consensus has yet to be achieved. In this report, we propose an in silico reference gene validation method, iRGvalid, that can be used as a universal tool to validate the reference genes recommended from different resources so as to identify the best ones without a need for any wet lab validation tests.MethodsiRGvalid takes advantage of high throughput gene expression data and is built on a double-normalization strategy. First, the expression level of each individual gene is normalized against the total gene expression level of each sample, followed by a target gene normalization to the candidate reference gene(s). Linear regression analysis is then performed between the pre- and post- normalized target gene across the whole sample set to evaluate the stability of the reference gene(s), which is positively associated with the Pearson correlation coefficient, Rt. The higher the Rt value, the more stable the reference gene. We applied iRGvalid to 14 candidate reference genes to validate and identify the most stable reference genes in four cancer types: lung adenocarcinoma, breast cancer, colon adenocarcinoma, and nasopharyngeal cancer. The stability of the reference gene is evaluated both individually and in groups of all possible combinations.ResultsHighly stable reference genes resulted in high Rt values regardless of the target gene used. The highest stability was achieved with a specific combination of 3 to 6 reference genes. A few genes were among the best reference genes across the cancer types studied here.ConclusioniRGvalid provides an easy and robust method to validate and identify the most stable reference gene or genes from a pool of candidate reference genes. The inclusivity of large expression data sets as well as the direct comparison of candidate reference genes makes it possible to identify reference genes with universal quality. This method can be used in any other gene expression studies when large cohorts of expression data are available.

The selection of suitable reference genes (RGs), especially the identification of the proper combination of RGs is the key to obtain reliable results of gene expression for quantitative real-time polymerase chain reaction (qRT-PCR). To date, there is no relevant study dealing with the stability of RGs in rat placenta. In this study, the geNorm, NormFinder, and BestKeeper software were used to analyze the expression stability of the candidate RGs in placenta under physiological and prenatal caffeine exposure (PCE) conditions. The expression of Tbp, Gapdh and Ywhaz in female and Polr2a, Gapdh and Ywhaz in male placenta were highly stable under physiological conditions, and there was no obvious gender difference. We further found that two RGs were sufficient for reliable normalization in female and male placenta and the combination of Ywhaz and Gapdh was the most suitable compound RGs under physiological conditions. Under PCE conditions, Ywhaz, Gapdh and Polr2a were the most stable genes in both female and male placenta. Among them, Ywhaz and Gapdh were chosen as the best paring. Finally, selected RGs were employed for normalization of the expression of a clear target gene and the results of standardization supported our choice. In conclusion, our study confirmed that Ywhaz/Gapdh combination was the most suitable RGs in rat placenta under physiological and PCE pathological conditions and provided a theoretical and experimental basis for physiological and pathological research of the rat placenta.

Background. Endocrine disrupting chemicals (EDCs) are natural and anthropogenic compounds discharged into the environment known to disrupt the endocrine system of humans and animals by mimicking functions of steroids in vivo. Many important events occurring during early postembryonic development, in relation to the gene expression attracted our attention. Quantitative real-time PCR (qRT-PCR) is a sensitive and highly reproducible method for gene expression analysis, with gene expression levels quantified by normalization to reference gene. The aim of this study was to select the suitable reference gene after EDCs exposure and during early postembryonic development. Materials and methods. For the study of the fish age effect, juveniles of Gobiocypris rarus Ye et Fu, 1983, were obtained at: 18, 22, 26, 30, 34, 38, 42, 46, and 50 days post fertilization (dpf). For mRNA expression analysis of the juvenile fish after EDCs treatment, the juveniles at 31 dpf were exposed to bisphenol A (BPA) (10 nM) and 17α-ethinylestradiol (EE2) (1 nM), respectively dissolved in dimethyl sulfoxide (DMSO) or solvent (0.001% DMSO, v/v) control group for 3 days. Cq values of the reference genes were obtained using qRT-PCR. The stability of these reference genes was analyzed by BestKeeper, geNorm, and NormFinder software, respectively. The expression of each reference gene was calculated using the 2–ΔCq method. In parallel, the mRNA expressions of cyp19a1b were normalized by the single most/least stable reference gene and the combinations of top-ranked reference genes.  Results. In this study, six candidate reference genes, actb, ef1a, gapdh, g6pd, tbp, and tuba1, were chosen to analyze their expression stability in relation to fish age and in the juvenile fish exposed to BPA and EE2. During early postembryonic development of Gobiocypris rarus, actb，ef1a, and gapdh were identified as the most stably expressed reference genes. In the juvenile fish exposed to BPA and EE2 for three days, gapdh, and actb were the most stable. However, g6pd and tuba1 were identified as the least stably expressed genes during the early postembryonic development and under BPA and EE2 exposure.  Conclusion. The presently reported study suggested that the mRNA expressions of the reference genes could be affected by chemical exposure or different physiological periods. In addition, it was indicated that stable reference gene should be selected to normalize the target gene expression to assure the correctness and accuracy of the experiment results. The last but not the least, we successfully obtained five commonly used reference genes of Gobiocypris rarus Ye et Fu, 1983, which can be applied in future studies serving as the stable reference gene and providing a broader range of selecting the stable reference gene.

BackgroundGene expression analysis using quantitative reverse transcription PCR (qRT-PCR) is a robust method wherein the expression levels of target genes are normalised using internal control genes, known as reference genes, to derive changes in gene expression levels. Although reference genes have recently been suggested for olive tissues, combined/independent analysis on different cultivars has not yet been tested. Therefore, an assessment of reference genes was required to validate the recent findings and select stably expressed genes across different olive cultivars.ResultsA total of eight candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine/threonine-protein phosphatase catalytic subunit (PP2A), elongation factor 1 alpha (EF1-alpha), polyubiquitin (OUB2), aquaporin tonoplast intrinsic protein (TIP2), tubulin alpha (TUBA), 60S ribosomal protein L18-3 (60S RBP L18-3) and polypyrimidine tract-binding protein homolog 3 (PTB)] were chosen based on their stability in olive tissues as well as in other plants. Expression stability was examined by qRT-PCR across 12 biological samples, representing mesocarp tissues at various developmental stages in three different olive cultivars, Barnea, Frantoio and Picual, independently and together during the 2009 season with two software programs, GeNorm and BestKeeper. Both software packages identified GAPDH, EF1-alpha and PP2A as the three most stable reference genes across the three cultivars and in the cultivar, Barnea. GAPDH, EF1-alpha and 60S RBP L18-3 were found to be most stable reference genes in the cultivar Frantoio while 60S RBP L18-3, OUB2 and PP2A were found to be most stable reference genes in the cultivar Picual.ConclusionsThe analyses of expression stability of reference genes using qRT-PCR revealed that GAPDH, EF1-alpha, PP2A, 60S RBP L18-3 and OUB2 are suitable reference genes for expression analysis in developing Olea europaea mesocarp tissues, displaying the highest level of expression stability across three different olive cultivars, Barnea, Frantoio and Picual, however the combination of the three most stable reference genes do vary amongst individual cultivars. This study will provide guidance to other researchers to select reference genes for normalization against target genes by qPCR across tissues obtained from the mesocarp region of the olive fruit in the cultivars, Barnea, Frantoio and Picual.

In vitro fertilization (IVF) is crucial for livestock reproduction; however, pregnancy rates after embryo transfer vary depending on the developmental speeds of the embryos. Although quantitative PCR (qPCR) is used to predict developmental potential, its reliability depends on the selection of appropriate reference genes (RGs) for normalization. To determine suitable RGs in bovine blastocysts with different developing speed, we evaluated the stability of eight candidate RGs (18S, ACTB, GAPDH, HMBS, PPIA, TBP, HPRT1, and SDHA) in early-, mid-, and late-developing IVF blastocysts (E-BL, M-BL, and L-BL, respectively) using RefFinder. Despite morphological similarities, E-BL, M-BL, and L-BL exhibited different biological features, including significantly lower pregnancy rates in L-BL than in the other groups, and less abundant transcript levels of five candidate RGs in L-BL than in E-BL. RefFinder revealed that ACTB was the most stable RG, whereas TBP was the least stable. To emphasize the critical importance of selecting stable RGs, we analyzed the expression of key developmental markers including those of the inner cell mass (ICM; OCT4, SOX2) and trophectoderm (TE; CDX2, GATA3, IFNτ), using various RGs for normalization. For ICM markers, normalization with ACTB showed results consistent with pregnancy rates, whereas moderately stable (18S) and less stable (TBP) RGs yielded contradictory outcomes. Normalization with unstable RGs produced inconsistent TE marker expression patterns (CDX2, GATA3) and overestimated (IFNτ) results across groups, compared with the results of ACTB. These results demonstrate that selecting inappropriate RGs for qPCR normalization can lead to misinterpretation, highlighting the necessity of proper RG evaluation to ensure accurate results in bovine embryo research.

The relationship between the conceptus and the maternal uterine environment is crucial for the successful establishment and maintenance of pregnancy in cattle. Gene expression analysis of the conceptus and maternal reproductive tissues is a favourable method to assess the embryonic maternal interaction. The reliability of the commonly used method reverse transcription-quantitative polymerase chain reaction (RT-qPCR) depends on proper normalization to stable reference genes (RGs). The objective of this study was to determine the expression stability of 10 potential RGs in maternal reproductive tissues and foetal tissues, and to analyse the effect of RG selection on the calculation of the relative expression of target genes. The expression stability of 10 potential RGs was analysed in eight different tissues from three pregnant dairy cows. Three programs-GeNorm, NormFinder and Bestkeeper-were used to identify the best RGs. According to all three programs, the most stable RG was CNOT11, whereas the least stable RGs were GAPDH and HPRT1. GeNorm analysis showed that a combination of five RGs (SDHA, PPIA, CNOT11, RPS9 and RPL19) was necessary for appropriate data normalization. However, NormFinder analysis indicated that the combination of CNOT11 and PPIA was the most suitable. When target genes were normalized to these RGs, the relative expression of the Radical S-adenosyl methionine domain containing 2 gene was not affected by the choice of RGs, whereas a large difference was observed in the expression profile of the Nuclear erythroid2-related factor 2 gene between the most stable and least stable RGs. The results indicate that careful selection of RGs is crucial under different conditions, especially for target genes with relatively small fold changes. Furthermore, the results provide useful information for the selection of RGs for evaluating genes affecting bovine reproduction.

Understanding the mechanism of seed germination requires a sturdy selection of reference genes for expression analysis using qRT-PCR. The accurate normalization of genes becomes necessary to circumvent erroneous result, as housekeeping genes does not remain stable over the different experimental condition in various tissue or species. Reliable and stable reference gene during ethrel induced germination of groundnut seeds is not reported yet. In this study, seven candidate reference genes were selected based on previous reports in groundnut under different experimental conditions. Seeds of NRCG 14380 (dormant) and TAG 24 (non-dormant) genotypes were treated with ethrel and sampled at 0, 6, 12 and 24 hours after incubation. The stability of reference genes such as actin1 (ACT1), actin11 (ACT11), alcohol dehydrogenase (ADH3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate dehydrogenase (G6PD), ubiquitin C (UBC) and α-Tubulin (TUA) was analyzed through a different statistical algorithm such as BestKeeper, NormFinder, geNorm and its consensus stability ranking was retrieved using RefFinder. Among all reference genes studied, ACT1 was found most stable reference gene in our study. Stability and reliability of ACT1 and ADH3 (most stable) reference genes were further validated through qRT- PCR with relative quantification of two germination related genes ACC oxidase1 (ACO1) and gibberellic acid regulated protein which showed consistency in their expression level. Our result revealed a stable reference gene that could be used as an internal control gene for gene expression study of ethrel treated groundnut seed germination.

Identification and validation of reference genes for RT-qPCR analysis in fetal rat pancreas

Selecting appropriate reference genes is vital to normalize gene expression analysis in birch (Betula platyphylla) under different abiotic stress conditions using quantitative real-time reverse transcription PCR (qRT-PCR). In this study, 11 candidate birch reference genes (ACT, TUA, TUB, TEF, 18S rRNA, EF1α, GAPDH, UBC, YLS8, SAND, and CDPK) were selected to evaluate the stability of their expression in different tissues and under different abiotic stress conditions. Three statistical algorithms (GeNorm, NormFinder, and BestKeeper) were used to analyze the stability of the 11 candidate reference genes to identify the most appropriate one. The results indicated that EF-1α was the most stable reference gene in different birch tissues, ACT was the most stable reference gene for normal conditions, ACT and TEF were the most stable reference genes for salt stress treatment, TUB was the most stable reference gene for osmotic stress treatment, and ACT was the most appropriate choice in all samples of birch. In conclusion, the most appropriate reference genes varied among different experimental conditions. However, in this study, ACT was the optimum reference gene in all experimental groups, except in the different tissues group. GAPDH was the least stable candidate reference gene in all experimental conditions. In addition, three stress-induced genes (BpGRAS1, BpGRAS16, and BpGRAS19) were chosen to verify the stability of the selected reference genes in different tissues and under salt stress. This study laid the foundation for the selection of appropriate reference gene(s) for future gene expression pattern studies in birch.

Selecting appropriate reference genes is vital to normalize gene expression analysis in birch (Betula platyphylla) under different abiotic stress conditions using quantitative real-time reverse transcription PCR (qRT-PCR). In this study, 11 candidate birch reference genes (ACT, TUA, TUB, TEF, 18S rRNA, EF1α, GAPDH, UBC, YLS8, SAND, and CDPK) were selected to evaluate the stability of their expression in different tissues and under different abiotic stress conditions. Three statistical algorithms (GeNorm, NormFinder, and BestKeeper) were used to analyze the stability of the 11 candidate reference genes to identify the most appropriate one. The results indicated that EF-1α was the most stable reference gene in different birch tissues, ACT was the most stable reference gene for normal conditions, ACT and TEF were the most stable reference genes for salt stress treatment, TUB was the most stable reference gene for osmotic stress treatment, and ACT was the most appropriate choice in all samples of birch. In conclusion, the most appropriate reference genes varied among different experimental conditions. However, in this study, ACT was the optimum reference gene in all experimental groups, except in the different tissues group. GAPDH was the least stable candidate reference gene in all experimental conditions. In addition, three stress-induced genes (BpGRAS1, BpGRAS16, and BpGRAS19) were chosen to verify the stability of the selected reference genes in different tissues and under salt stress. This study laid the foundation for the selection of appropriate reference gene(s) for future gene expression pattern studies in birch.

Evaluation of internal reference genes in Auxenochlorella protothecoides under continuous heterotrophic culture conditions at normal, low and high temperatures