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Sample records for qpcr reference genes

  1. Reference Gene Selection for qPCR Normalization of Kosteletzkya virginica under Salt Stress

    PubMed Central

    Tang, Xiaoli; Wang, Hongyan; Shao, Chuyang; Shao, Hongbo

    2015-01-01

    Kosteletzkya virginica (L.) is a newly introduced perennial halophytic plant. Presently, reverse transcription quantitative real-time PCR (qPCR) is regarded as the best choice for analyzing gene expression and its accuracy mainly depends on the reference genes which are used for gene expression normalization. In this study, we employed qPCR to select the most stable reference gene in K. virginica which showed stable expression profiles under our experimental conditions. The candidate reference genes were 18S ribosomal RNA (18SrRNA), β-actin (ACT), α-tubulin (TUA), and elongation factor (EF). We tracked the gene expression profiles of the candidate genes and analyzed their stabilities through BestKeeper, geNorm, and NormFinder software programs. The results of the three programs were identical and 18SrRNA was assessed to be the most stable reference gene in this study. However, TUA was identified to be the most unstable. Our study proved again that the traditional reference genes indeed displayed a certain degree of variations under given experimental conditions. Importantly, our research also provides guidance for selecting most suitable reference genes and lays the foundation for further studies in K. virginica. PMID:26581422

  2. Optimal reference genes for qPCR in resting and activated human NK cells--Flow cytometric data correspond to qPCR gene expression analysis.

    PubMed

    Kaszubowska, Lucyna; Wierzbicki, Piotr Mieczysław; Karsznia, Sylwia; Damska, Marta; Ślebioda, Tomasz Jerzy; Foerster, Jerzy; Kmieć, Zbigniew

    2015-07-01

    Natural killer cells (NK cells) are cytotoxic lymphocytes critical to the innate immune system engaged in rapid response against tumor or virus infected cells. After activation NK cells acquire enhanced cytotoxicity and are capable of producing cytokines to stimulate other immune cells. Quantitative PCR (qPCR) is a method of choice for gene expression analysis but the usage of reliable reference genes for the normalization process is critical. Commonly used reference genes may vary in expression level between different experimental conditions providing wrong quantitative results of the studied genes' expression levels. Fourteen potential endogenous control genes were analyzed by qPCR method in NK-92 cell line that shows characteristics of human natural killer cells and is often used in studies on biology of NK lymphocytes. NK-92 cells were stimulated with IL-2 or TNF for 2, 24 or 72 h. Results were analyzed with RefFinder, a program which enables evaluation and screening of reference genes and integrates the currently available major computational programs (Genorm, Normfinder, BestKeeper and Delta Ct). The most stable gene in activated and non-activated NK cells was B2M, followed by IPO-8 and GAPDH and the least stable were HPRT1, PPIA and RPL32. The normalization process was performed on SOD2 gene and the results of qPCR experiments were confirmed by flow cytometry. The flow cytometric data corresponded to the results of qPCR gene expression analysis performed for the reference genes qualified by RefFinder as the most stable.

  3. Selection of reference genes for gene expression studies in zucchini (Cucurbita pepo) using qPCR.

    PubMed

    Obrero, Angeles; Die, Jose V; Román, Belén; Gómez, Pedro; Nadal, Salvador; González-Verdejo, Clara I

    2011-05-25

    The zucchini (Cucurbita pepo) is an important food crop, the transcriptomics of which are a fundamental tool to accelerate the development of new varieties by breeders. However, the suitability of reference genes for data normalization in zucchini has not yet been studied. The aim of this study was to assess the suitability of 13 genes for their potential use as reference genes in quantitative real-time PCR. Assays were performed on 34 cDNA samples representing plants under different stresses and at different developmental stages. The application of geNorm and NormFinder software revealed that the use of a combination of UFP, EF-1A, RPL36aA, PP2A, and CAC genes for the different experimental sets was the best strategy for reliable normalization. In contrast, 18S rRNA and TUA were less stable and unsuitable for use as internal controls. These results provide the possibility to allow more accurate use of qPCR in this horticultural crop.

  4. Selection of reference genes for qPCR in hairy root cultures of peanut

    PubMed Central

    2011-01-01

    Background Hairy root cultures produced via Agrobacterium rhizogenes-mediated transformation have emerged as practical biological models to elucidate the biosynthesis of specialized metabolites. To effectively understand the expression patterns of the genes involved in the metabolic pathways of these compounds, reference genes need to be systematically validated under specific experimental conditions as established by the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. In the present report we describe the first validation of reference genes for RT-qPCR in hairy root cultures of peanut which produce stilbenoids upon elicitor treatments. Results A total of 21 candidate reference genes were evaluated. Nineteen genes were selected based on previous qPCR studies in plants and two were from the T-DNAs transferred from A. rhizogenes. Nucleotide sequences of peanut candidate genes were obtained using their homologous sequences in Arabidopsis. To identify the suitable primers, calibration curves were obtained for each candidate reference gene. After data analysis, 12 candidate genes meeting standard efficiency criteria were selected. The expression stability of these genes was analyzed using geNorm and NormFinder algorithms and a ranking was established based on expression stability of the genes. Candidate reference gene expression was shown to have less variation in methyl jasmonate (MeJA) treated root cultures than those treated with sodium acetate (NaOAc). Conclusions This work constitutes the first effort to validate reference genes for RT-qPCR in hairy roots. While these genes were selected under conditions of NaOAc and MeJA treatment, we anticipate these genes to provide good targets for reference genes for hairy roots under a variety of stress conditions. The lead reference genes were a gene encoding for a TATA box binding protein (TBP2) and a gene encoding a ribosomal protein (RPL8C). A commonly used reference gene

  5. Validation of qPCR reference genes in lymphocytes from patients with amyotrophic lateral sclerosis

    PubMed Central

    Usarek, Ewa; Barańczyk-Kuźma, Anna; Kaźmierczak, Beata; Gajewska, Beata; Kuźma-Kozakiewicz, Magdalena

    2017-01-01

    Quantitative polymerase chain reaction (qPCR) is the most specific and reliable method for determination of mRNA gene expression. Crucial point for its accurate normalization is the choice of appropriate internal control genes (ICGs). In the present work we determined and compare the expression of eight commonly used ICGs in lymphocytes from 26 patients with amyotrophic lateral sclerosis (ALS) and 30 control subjects. Peripheral blood mononuclear cells (PBMCs) before and after immortalization by EBV transfection (lymphoblast cell lines—LCLs) were used for qPCR analysis. LCLs were studied before and after liquid nitrogen cryopreservation and culturing (groups LCL1 and LCL2, respectively). qPCR data of 8 ICGs expression was analyzed by BestKeeper, NormFinder and geNorm methods. All studied genes (18SRNA, ACTB, B2M, GUSB,GAPDH, HPRT1, MT-ATP6 and RPS17) were expressed in PBMCs, whereas only first four in LCLs. LCLs cryopreservation had no effect on ICGs expression. Comprehensive ranking indicated RPS17 with MT-ATP6 as the best ICGs for qPCR in PBMCs of control and ALS subjects, and RPS17 with 18RNA or MT-ATP6 in LCLs from ALS. In PBMCs 18RNA shouldn’t be used as ICG. PMID:28328930

  6. The utility of optical detection system (qPCR) and bioinformatics methods in reference gene expression analysis

    NASA Astrophysics Data System (ADS)

    Skarzyńska, Agnieszka; Pawełkowicz, Magdalena; PlÄ der, Wojciech; Przybecki, Zbigniew

    2016-09-01

    Real-time quantitative polymerase chain reaction is consider as the most reliable method for gene expression studies. However, the expression of target gene could be misinterpreted due to improper normalization. Therefore, the crucial step for analysing of qPCR data is selection of suitable reference genes, which should be validated experimentally. In order to choice the gene with stable expression in the designed experiment, we performed reference gene expression analysis. In this study genes described in the literature and novel genes predicted as control genes, based on the in silico analysis of transcriptome data were used. Analysis with geNorm and NormFinder algorithms allow to create the ranking of candidate genes and indicate the best reference for flower morphogenesis study. According to the results, genes CACS and CYCL were characterised the most stable expression, but the least suitable genes were TUA and EF.

  7. Evaluation of candidate reference genes for QPCR during ontogenesis and of immune-relevant tissues of European seabass (Dicentrarchus labrax).

    PubMed

    Mitter, Karin; Kotoulas, Georgios; Magoulas, Antonios; Mulero, Victor; Sepulcre, Pilar; Figueras, Antonio; Novoa, Beatrice; Sarropoulou, Elena

    2009-08-01

    The expression level of mRNA can vary significantly in different experimental conditions, such as stress, infection, developmental stage or tissue. Suitable reference genes are expected to exhibit constant expression levels. However no single gene is constitutively expressed in all cell types and under all experimental conditions. It has become clear that expression stability of the intended reference gene has to be examined before each experiment. For expression studies using quantitative real-time PCR (qPCR) at least two reference genes have to be applied. So far expression studies in the European seabass (Dicentrarchus labrax) as well as in the Gilthead seabream (Sparus aurata) have been performed with only one reference gene (S18, Ef-1 alpha or Gapdh). Though significant variations showed up in other teleost species such as the Atlantic halibut and the zebrafish affirming the need for proper normalization strategies, the present study aims at identifying suitable reference genes among nine candidates [glyceraldehyde-phosphate-dehydrogenase (Gapdh), beta-actin (two regions of beta-actin), 40S ribosomal protein S30 (Fau), ribosomal protein L13 a (L13a), beta2-tubulin (Tubb2) and tyrosine 3 monooxygenase/tryptophan 5-monooxygenase activation protein (Tyr)] for expression analysis of 8 developmental stages and a tissue panel (spleen, liver, kidney and brain) with samples infected with Nodavirus and Vibrio anguillarum in D. labrax. Besides the analysis of raw Ct-values, the gene expression stability was determined using two different software applications BestKeeper and NormFinder. According to both algorithms the best two reference genes for an appropriate normalization approach during D. labrax development are Ef-1 alpha and L13a whereas in the tissue panel Fau and L13a are recommended for qPCR normalization.

  8. Validation and Comparison of Reference Genes for qPCR Normalization of Celery (Apium graveolens) at Different Development Stages.

    PubMed

    Li, Meng-Yao; Wang, Feng; Jiang, Qian; Wang, Guan-Long; Tian, Chang; Xiong, Ai-Sheng

    2016-01-01

    A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in qPCR analysis. Celery is one of the representative vegetable in Apiaceae and is widely cultivated and consumed in the world. However, no reports have been previously published concerning reference genes in celery. In this study, the expression stabilities of nine candidate reference genes in leaf blade and petiole at different development stages were evaluated using three statistics algorithms geNorm, NormFinder, and BestKeeper. Our results showed that TUB-B, TUB-A, and UBC were the most reference genes among all tested samples. GAPDH represented the maximum stability for most individual sample, while the UBQ displayed the minimum stability. To further validate the stability of reference genes, the expression pattern of AgAP2-2 was calculated by using the selected genes for normalization. In addition, the expression patterns of several development-related genes were studied using the selected reference gene. Our results will be beneficial for further studies on gene transcription in celery.

  9. Evaluation of Suitable Reference Genes for Normalization of qPCR Gene Expression Studies in Brinjal (Solanum melongena L.) During Fruit Developmental Stages.

    PubMed

    Kanakachari, Mogilicherla; Solanke, Amolkumar U; Prabhakaran, Narayanasamy; Ahmad, Israr; Dhandapani, Gurusamy; Jayabalan, Narayanasamy; Kumar, Polumetla Ananda

    2016-02-01

    Brinjal/eggplant/aubergine is one of the major solanaceous vegetable crops. Recent availability of genome information greatly facilitates the fundamental research on brinjal. Gene expression patterns during different stages of fruit development can provide clues towards the understanding of its biological functions. Quantitative real-time PCR (qPCR) has become one of the most widely used methods for rapid and accurate quantification of gene expression. However, its success depends on the use of a suitable reference gene for data normalization. For qPCR analysis, a single reference gene is not universally suitable for all experiments. Therefore, reference gene validation is a crucial step. Suitable reference genes for qPCR analysis of brinjal fruit development have not been investigated so far. In this study, we have selected 21 candidate reference genes from the Brinjal (Solanum melongena) Plant Gene Indices database (compbio.dfci.harvard.edu/tgi/plant.html) and studied their expression profiles by qPCR during six different fruit developmental stages (0, 5, 10, 20, 30, and 50 days post anthesis) along with leaf samples of the Pusa Purple Long (PPL) variety. To evaluate the stability of gene expression, geNorm and NormFinder analytical softwares were used. geNorm identified SAND (SAND family protein) and TBP (TATA binding protein) as the best pairs of reference genes in brinjal fruit development. The results showed that for brinjal fruit development, individual or a combination of reference genes should be selected for data normalization. NormFinder identified Expressed gene (expressed sequence) as the best single reference gene in brinjal fruit development. In this study, we have identified and validated for the first time reference genes to provide accurate transcript normalization and quantification at various fruit developmental stages of brinjal which can also be useful for gene expression studies in other Solanaceae plant species.

  10. Characterization of reference genes for qPCR analysis in various tissues of the Fujian oyster Crassostrea angulata

    NASA Astrophysics Data System (ADS)

    Pu, Fei; Yang, Bingye; Ke, Caihuan

    2015-07-01

    Accurate quantification of transcripts using quantitative real-time polymerase chain reaction (qPCR) depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2 ( ACT-2), elongation factor 1 alpha ( EF-1α), elongation factor 1 beta ( EF-1β), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH), ubiquitin ( UBQ), β-tubulin ( β-TUB), and 18S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using qPCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the geNorm software to assess the expression stability of the candidate reference genes, revealing that β-TUB and UBQ were the most stable genes. The commonly used GAPDH and 18S rRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene ( Gβ) across tissue types was also examined and normalized to the expression of each or both of UBQ and β-TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β-TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in Crassostrea angulata, which will be indispensable for further functional genomics studies in this economically valuable marine bivalve.

  11. Identification of reference genes for qPCR analysis during hASC long culture maintenance

    PubMed Central

    Palombella, Silvia; Pirrone, Cristina; Cherubino, Mario; Valdatta, Luigi; Bernardini, Giovanni

    2017-01-01

    Up to now quantitative PCR based assay is the most common method for characterizing or confirming gene expression patterns and comparing mRNA levels in different sample populations. Since this technique is relative easy and low cost compared to other methods of characterization, e.g. flow cytometry, we used it to typify human adipose-derived stem cells (hASCs). hASCs possess several characteristics that make them attractive for scientific research and clinical applications. Accurate normalization of gene expression relies on good selection of reference genes and the best way to choose them appropriately is to follow the common rule of the “Best 3”, at least three reference genes, three different validation software and three sample replicates. Analysis was performed on hASCs cultivated until the eleventh cell confluence using twelve candidate reference genes, initially selected from literature, whose stability was evaluated by the algorithms NormFinder, BestKeeper, RefFinder and IdealRef, a home-made version of GeNorm. The best gene panel (RPL13A, RPS18, GAPDH, B2M, PPIA and ACTB), determined in one patient by IdealRef calculation, was then investigated in other four donors. Although patients demonstrated a certain gene expression variability, we can assert that ACTB is the most unreliable gene whereas ribosomal proteins (RPL13A and RPS18) show minor inconstancy in their mRNA expression. This work underlines the importance of validating reference genes before conducting each experiment and proposes a free software as alternative to those existing. PMID:28182697

  12. A Panel of Stably Expressed Reference Genes for Real-Time qPCR Gene Expression Studies of Mallards (Anas platyrhynchos).

    PubMed

    Chapman, Joanne R; Helin, Anu S; Wille, Michelle; Atterby, Clara; Järhult, Josef D; Fridlund, Jimmy S; Waldenström, Jonas

    2016-01-01

    Determining which reference genes have the highest stability, and are therefore appropriate for normalising data, is a crucial step in the design of real-time quantitative PCR (qPCR) gene expression studies. This is particularly warranted in non-model and ecologically important species for which appropriate reference genes are lacking, such as the mallard--a key reservoir of many diseases with relevance for human and livestock health. Previous studies assessing gene expression changes as a consequence of infection in mallards have nearly universally used β-actin and/or GAPDH as reference genes without confirming their suitability as normalisers. The use of reference genes at random, without regard for stability of expression across treatment groups, can result in erroneous interpretation of data. Here, eleven putative reference genes for use in gene expression studies of the mallard were evaluated, across six different tissues, using a low pathogenic avian influenza A virus infection model. Tissue type influenced the selection of reference genes, whereby different genes were stable in blood, spleen, lung, gastrointestinal tract and colon. β-actin and GAPDH generally displayed low stability and are therefore inappropriate reference genes in many cases. The use of different algorithms (GeNorm and NormFinder) affected stability rankings, but for both algorithms it was possible to find a combination of two stable reference genes with which to normalise qPCR data in mallards. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies in ducks. The fact that nearly all previous studies of the influence of pathogen infection on mallard gene expression have used a single, non-validated reference gene is problematic. The toolkit of putative reference genes provided here offers a solid foundation for future studies of gene expression in mallards and other waterfowl.

  13. Comparative Validation of Conventional and RNA-Seq Data-Derived Reference Genes for qPCR Expression Studies of Colletotrichum kahawae

    PubMed Central

    Vieira, Ana; Cabral, Ana; Fino, Joana; Azinheira, Helena G.; Loureiro, Andreia; Talhinhas, Pedro; Pires, Ana Sofia; Varzea, Vitor; Moncada, Pilar; Oliveira, Helena; Silva, Maria do Céu; Paulo, Octávio S.; Batista, Dora

    2016-01-01

    Colletotrichum kahawae is an emergent fungal pathogen causing severe epidemics of Coffee Berry Disease on Arabica coffee crops in Africa. Currently, the molecular mechanisms underlying the Coffea arabica—C. kahawae interaction are still poorly understood, as well as the differences in pathogen aggressiveness, which makes the development of functional studies for this pathosystem a crucial step. Quantitative real time PCR (qPCR) has been one of the most promising approaches to perform gene expression analyses. However, proper data normalization with suitable reference genes is an absolute requirement. In this study, a set of 8 candidate reference genes were selected based on two different approaches (literature and Illumina RNA-seq datasets) to assess the best normalization factor for qPCR expression analysis of C. kahawae samples. The gene expression stability of candidate reference genes was evaluated for four isolates of C. kahawae bearing different aggressiveness patterns (Ang29, Ang67, Zim12 and Que2), at different stages of fungal development and key time points of the plant-fungus interaction process. Gene expression stability was assessed using the pairwise method incorporated in geNorm and the model-based method used by NormFinder software. For C. arabica—C. kahawae interaction samples, the best normalization factor included the combination of PP1, Act and ck34620 genes, while for C. kahawae samples the combination of PP1, Act and ck20430 revealed to be the most appropriate choice. These results suggest that RNA-seq analyses can provide alternative sources of reference genes in addition to classical reference genes. The analysis of expression profiles of bifunctional catalase-peroxidase (cat2) and trihydroxynaphthalene reductase (thr1) genes further enabled the validation of the selected reference genes. This study provides, for the first time, the tools required to conduct accurate qPCR studies in C. kahawae considering its aggressiveness pattern

  14. Reference Gene Selection for qPCR Analysis in Tomato-Bipartite Begomovirus Interaction and Validation in Additional Tomato-Virus Pathosystems

    PubMed Central

    Lacerda, Ana L. M.; Fonseca, Leonardo N.; Blawid, Rosana; Boiteux, Leonardo S.; Ribeiro, Simone G.; Brasileiro, Ana C. M.

    2015-01-01

    Quantitative Polymerase Chain Reaction (qPCR) is currently the most sensitive technique used for absolute and relative quantification of a target gene transcript, requiring the use of appropriated reference genes for data normalization. To accurately estimate the relative expression of target tomato (Solanum lycopersicum L.) genes responsive to several virus species in reverse transcription qPCR analysis, the identification of reliable reference genes is mandatory. In the present study, ten reference genes were analyzed across a set of eight samples: two tomato contrasting genotypes (‘Santa Clara’, susceptible, and its near-isogenic line ‘LAM 157’, resistant); subjected to two treatments (inoculation with Tomato chlorotic mottle virus (ToCMoV) and its mock-inoculated control) and in two distinct times after inoculation (early and late). Reference genes stability was estimated by three statistical programs (geNorm, NormFinder and BestKeeper). To validate the results over broader experimental conditions, a set of ten samples, corresponding to additional three tomato-virus pathosystems that included tospovirus, crinivirus and tymovirus + tobamovirus, was analyzed together with the tomato-ToCMoV pathosystem dataset, using the same algorithms. Taking into account the combined analyses of the ranking order outputs from the three algorithms, TIP41 and EF1 were identified as the most stable genes for tomato-ToCMoV pathosystem, and TIP41 and EXP for the four pathosystems together, and selected to be used as reference in the forthcoming expression qPCR analysis of target genes in experimental conditions involving the aforementioned tomato-virus pathosystems. PMID:26317870

  15. Validation of Reference Genes for Real-Time Quantitative PCR (qPCR) Analysis of Avibacterium paragallinarum

    PubMed Central

    Wen, Shuxiang; Chen, Xiaoling; Xu, Fuzhou; Sun, Huiling

    2016-01-01

    Real-time quantitative reverse transcription PCR (qRT-PCR) offers a robust method for measurement of gene expression levels. Selection of reliable reference gene(s) for gene expression study is conducive to reduce variations derived from different amounts of RNA and cDNA, the efficiency of the reverse transcriptase or polymerase enzymes. Until now reference genes identified for other members of the family Pasteurellaceae have not been validated for Avibacterium paragallinarum. The aim of this study was to validate nine reference genes of serovars A, B, and C strains of A. paragallinarum in different growth phase by qRT-PCR. Three of the most widely used statistical algorithms, geNorm, NormFinder and ΔCT method were used to evaluate the expression stability of reference genes. Data analyzed by overall rankings showed that in exponential and stationary phase of serovar A, the most stable reference genes were gyrA and atpD respectively; in exponential and stationary phase of serovar B, the most stable reference genes were atpD and recN respectively; in exponential and stationary phase of serovar C, the most stable reference genes were rpoB and recN respectively. This study provides recommendations for stable endogenous control genes for use in further studies involving measurement of gene expression levels. PMID:27942007

  16. Reference genes for real-time qPCR in leukocytes from asthmatic patients before and after anti-asthma treatment.

    PubMed

    Kozmus, Carina E P; Potočnik, Uroš

    2015-10-01

    The aim of this study was to develop a set of reference genes whose expression is stable and suitable for normalization of target gene expression measured in asthma patients during anti-asthmatic treatment. Real-time qPCR was used to determine expression of 7 candidate reference genes (18S rRNA, ACTB, B2M, GAPDH, POLR2A, RPL13A and RPL32) and 7 target genes in leukocytes from asthma patients before and after treatment with inhaled corticosteroids and leukotriene receptor antagonist. Variance of Cq values was analyzed and stability ranking was determined with geNorm. We further investigated how the different normalization strategies affected the consistency of conclusions if the specific investigated target gene is down-regulated or up-regulated after anti-asthmatic therapy. The top-ranking reference genes determined by geNorm, when samples before and after therapy were analyzed (ACTB, B2M and GAPDH) were different from those (POLR2A and B2M) when only samples before treatment were analyzed. Using only a single reference gene for normalization of 7 target gene expression compared to our strategy, there would be as low as 19% of consistency in conclusions. We suggest the use of the geometric mean of ACTB, B2M and GAPDH for normalization of qPCR data of target genes in pharmacogenomics studies in asthma patients before and after anti-asthmatic therapy, however if gene expression is measured only before anti-asthmatic treatment, we recommend the use of the geometric mean of POLR2A and B2M.

  17. LEMming: A Linear Error Model to Normalize Parallel Quantitative Real-Time PCR (qPCR) Data as an Alternative to Reference Gene Based Methods

    PubMed Central

    Feuer, Ronny; Vlaic, Sebastian; Arlt, Janine; Sawodny, Oliver; Dahmen, Uta; Zanger, Ulrich M.; Thomas, Maria

    2015-01-01

    Background Gene expression analysis is an essential part of biological and medical investigations. Quantitative real-time PCR (qPCR) is characterized with excellent sensitivity, dynamic range, reproducibility and is still regarded to be the gold standard for quantifying transcripts abundance. Parallelization of qPCR such as by microfluidic Taqman Fluidigm Biomark Platform enables evaluation of multiple transcripts in samples treated under various conditions. Despite advanced technologies, correct evaluation of the measurements remains challenging. Most widely used methods for evaluating or calculating gene expression data include geNorm and ΔΔCt, respectively. They rely on one or several stable reference genes (RGs) for normalization, thus potentially causing biased results. We therefore applied multivariable regression with a tailored error model to overcome the necessity of stable RGs. Results We developed a RG independent data normalization approach based on a tailored linear error model for parallel qPCR data, called LEMming. It uses the assumption that the mean Ct values within samples of similarly treated groups are equal. Performance of LEMming was evaluated in three data sets with different stability patterns of RGs and compared to the results of geNorm normalization. Data set 1 showed that both methods gave similar results if stable RGs are available. Data set 2 included RGs which are stable according to geNorm criteria, but became differentially expressed in normalized data evaluated by a t-test. geNorm-normalized data showed an effect of a shifted mean per gene per condition whereas LEMming-normalized data did not. Comparing the decrease of standard deviation from raw data to geNorm and to LEMming, the latter was superior. In data set 3 according to geNorm calculated average expression stability and pairwise variation, stable RGs were available, but t-tests of raw data contradicted this. Normalization with RGs resulted in distorted data contradicting

  18. Effect of irradiation on the expression of DNA repair genes studied in human fibroblasts by real-time qPCR using three methods of reference gene validation.

    PubMed

    Reuther, Sebastian; Reiter, Martina; Raabe, Annette; Dikomey, Ekkehard

    2013-11-01

    The aim of this study was to determine the effects of ionizing radiation on gene expression by using for a first time a qPCR platform specifically established for the detection of 94 DNA repair genes but also to test the robustness of these results by using three analytical methods (global pattern recognition, ΔΔCq/Normfinder and ΔΔCq/Genorm). Study was focused on these genes because DNA repair is known primarily to determine the radiation response. Six strains of normal human fibroblasts were exposed to 2 Gy, and changes in gene expression were analyzed 24 h thereafter. A significant change in gene expression was found for only few genes, but the genes detected were mostly different for the three analytical methods used. For GPR, a significant change was found for four genes, in contrast to the eight or nine genes when applying ΔΔCq/Genorm or ΔΔCq/Normfinder, respectively. When using all three methods, a significant change in expression was only seen for GADD45A and PCNA. These data demonstrate that (1) the genes identified to show an altered expression upon irradiation strongly depend on the analytical method applied, and that (2) overall GADD45A and PCNA appear to play a central role in this response, while no significant change is induced for any of the other DNA repair genes tested.

  19. RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR

    PubMed Central

    Gentile, Adriana-Mariel; Lhamyani, Said; Coín-Aragüez, Leticia; Oliva-Olivera, Wilfredo; Zayed, Hatem; Vega-Rioja, Antonio; Monteseirin, Javier; Romero-Zerbo, Silvana-Yanina; Tinahones, Francisco-José; Bermúdez-Silva, Francisco-Javier; El Bekay, Rajaa

    2016-01-01

    Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR. PMID:27304673

  20. Selection of reference genes for qPCR- and ddPCR-based analyses of gene expression in Senescing Barley leaves.

    PubMed

    Zmienko, Agnieszka; Samelak-Czajka, Anna; Goralski, Michal; Sobieszczuk-Nowicka, Ewa; Kozlowski, Piotr; Figlerowicz, Marek

    2015-01-01

    Leaf senescence is a tightly regulated developmental or stress-induced process. It is accompanied by dramatic changes in cell metabolism and structure, eventually leading to the disintegration of chloroplasts, the breakdown of leaf proteins, internucleosomal fragmentation of nuclear DNA and ultimately cell death. In light of the global and intense reorganization of the senescing leaf transcriptome, measuring time-course gene expression patterns in this model is challenging due to the evident problems associated with selecting stable reference genes. We have used oligonucleotide microarray data to identify 181 genes with stable expression in the course of dark-induced senescence of barley leaf. From those genes, we selected 5 candidates and confirmed their invariant expression by both reverse transcription quantitative PCR and droplet digital PCR (ddPCR). We used the selected reference genes to normalize the level of the expression of the following senescence-responsive genes in ddPCR assays: SAG12, ICL, AGXT, CS and RbcS. We were thereby able to achieve a substantial reduction in the data variability. Although the use of reference genes is not considered mandatory in ddPCR assays, our results show that it is advisable in special cases, specifically those that involve the following conditions: i) a low number of repeats, ii) the detection of low-fold changes in gene expression or iii) series data comparisons (such as time-course experiments) in which large sample variation greatly affects the overall gene expression profile and biological interpretation of the data.

  1. Validation of reference genes for normalization of qPCR mRNA expression levels in Staphylococcus aureus exposed to osmotic and lactic acid stress conditions encountered during food production and preservation.

    PubMed

    Sihto, Henna-Maria; Tasara, Taurai; Stephan, Roger; Johler, Sophia

    2014-07-01

    Staphylococcus aureus represents the most prevalent cause of food-borne intoxications worldwide. While being repressed by competing bacteria in most matrices, this pathogen exhibits crucial competitive advantages during growth at high salt concentrations or low pH, conditions frequently encountered in food production and preservation. We aimed to identify reference genes that could be used to normalize qPCR mRNA expression levels during growth of S. aureus in food-related osmotic (NaCl) and acidic (lactic acid) stress adaptation models. Expression stability of nine housekeeping genes was evaluated in full (LB) and nutrient-deficient (CYGP w/o glucose) medium under conditions of osmotic (4.5% NaCl) and acidic stress (lactic acid, pH 6.0) after 2-h exposure. Among the set of candidate reference genes investigated, rplD, rpoB,gyrB, and rho were most stably expressed in LB and thus represent the most suitable reference genes for normalization of qPCR data in osmotic or lactic acid stress models in a rich medium. Under nutrient-deficient conditions, expression of rho and rpoB was highly stable across all tested conditions. The presented comprehensive data on changes in expression of various S. aureus housekeeping genes under conditions of osmotic and lactic acid stress facilitate selection of reference genes for qPCR-based stress response models.

  2. Reference gene validation for qPCR on normoxia- and hypoxia-cultured human dermal fibroblasts exposed to UVA: is β-actin a reliable normalizer for photoaging studies?

    PubMed

    Brugè, F; Venditti, E; Tiano, L; Littarru, G P; Damiani, E

    2011-12-10

    Data normalization of gene expression on human dermal fibroblasts (HDF) exposed to UVA has commonly been done using either GAPDH or β-actin as reference genes without any validation of their expression stability. Since this aspect, important for accurate normalization, has been overlooked, we aimed to establish a suitable set of reference genes for studies on UVA-treated HDF cultured under both standard atmospheric oxygen tension (normoxia, 21%) and under a physiological, low oxygen tension for these cells (hypoxia, 5%). The stability of six commonly used reference genes was assessed using the geNorm and NormFinder softwares subsequent to reverse-transcription quantitative real-time PCR (RT-qPCR). GAPDH/SDHA were found to be the most stable genes under normoxia, while SDHA/TBP or HPRT1/β2M were the most stable ones under hypoxia in HDF exposed to 18 J/cm(2) UVA. β-Actin was always the most unstable reference gene. To emphasize the importance of selecting the most stably expressed reference genes for obtaining reliable results, mRNA expression levels of MMP-1 and COL1A1 were analyzed vs the best reference genes and the worst one. These reference genes are hence recommended for future qPCR analyses in studies concerning photo-damage on UVA-treated HDF.

  3. Reference Genes for qPCR Analysis in Resin-Tapped Adult Slash Pine As a Tool to Address the Molecular Basis of Commercial Resinosis

    PubMed Central

    de Lima, Júlio C.; de Costa, Fernanda; Füller, Thanise N.; Rodrigues-Corrêa, Kelly C. da Silva; Kerber, Magnus R.; Lima, Mariano S.; Fett, Janette P.; Fett-Neto, Arthur G.

    2016-01-01

    Pine oleoresin is a major source of terpenes, consisting of turpentine (mono- and sesquiterpenes) and rosin (diterpenes) fractions. Higher oleoresin yields are of economic interest, since oleoresin derivatives make up a valuable source of materials for chemical industries. Oleoresin can be extracted from living trees, often by the bark streak method, in which bark removal is done periodically, followed by application of stimulant paste containing sulfuric acid and other chemicals on the freshly wounded exposed surface. To better understand the molecular basis of chemically-stimulated and wound induced oleoresin production, we evaluated the stability of 11 putative reference genes for the purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis. Samples for RNA extraction were collected from field-grown adult trees under tapping operations using stimulant pastes with different compositions and at various time points after paste application. Statistical methods established by geNorm, NormFinder, and BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3 and UBI. To confirm expression stability of the candidate reference genes, expression profiles of putative P. elliottii orthologs of resin biosynthesis-related genes encoding Pinus contorta β-pinene synthase [PcTPS-(−)β-pin1], P. contorta levopimaradiene/abietadiene synthase (PcLAS1), Pinus taeda α-pinene synthase [PtTPS-(+)αpin], and P. taeda α-farnesene synthase (PtαFS) were examined following stimulant paste application. Increased oleoresin yields observed in stimulated treatments using phytohormone-based pastes were consistent with higher expression of pinene synthases. Overall, the expression of all genes examined matched the expected profiles of oleoresin-related transcript changes reported for previously examined conifers. PMID:27379135

  4. qPCR assays to quantify genes and gene expression associated with microbial perchlorate reduction.

    PubMed

    De Long, Susan K; Kinney, Kerry A; Kirisits, Mary Jo

    2010-11-01

    Quantitative PCR (qPCR) assays targeting cld (developed in this work) and pcrA (previously described) were used to quantify these perchlorate-related genes in a perchlorate-reducing enrichment culture. Transcript copies were quantified in perchlorate-reducing Rhodocyclaceae strain JDS4. Oxygen and nitrate inhibited expression of cld and pcrA.

  5. Comparison of TaqMan and SYBR Green qPCR methods for quantitative gene expression in tung tree tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative real-time-PCR (qPCR) is widely used for gene expression analysis due to its large dynamic range, tremendous sensitivity, high sequence-specificity, little to no post-amplification processing, and sample throughput. TaqMan and SYBR Green qPCR are two frequently used methods. However, dir...

  6. Comparison of TaqMan and SYBR Green qPCR methods for quantitative gene expression in tung tree tissues.

    PubMed

    Cao, Heping; Shockey, Jay M

    2012-12-19

    Quantitative real-time-PCR (qPCR) is widely used for gene expression analysis due to its large dynamic range, tremendous sensitivity, high sequence specificity, little to no postamplification processing, and sample throughput. TaqMan and SYBR Green qPCR are two frequently used methods. However, direct comparison of both methods using the same primers and biological samples is still limited. We compared both assays using seven RNAs from the seeds, leaves, and flowers of tung tree (Vernicia fordii), which produces high-value industrial oil. High-quality RNA were isolated from tung tissues, as indicated by a high rRNA ratio and RNA integrity number. qPCR primers and TaqMan probes were optimized. Under optimized conditions, both qPCR gave high correlation coefficiency and similar amplification efficiency, but TaqMan qPCR generated higher y-intercepts than SYBR Green qPCR, which overestimated the expression levels regardless of the genes and tissues tested. This is validated using well-known Dgat2 and Fadx gene expression in tung tissues. The results demonstrate that both assays are reliable for determining gene expression in tung tissues and that the TaqMan assay is more sensitive but generates lower calculated expression levels than the SYBR Green assay. This study suggests that any discussion of gene expression levels needs to be linked to which qPCR method is used in the analysis.

  7. A Whole-Transcriptome Approach to Evaluating Reference Genes for Quantitative Gene Expression Studies: A Case Study in Mimulus

    PubMed Central

    Stanton, Kimmy A.; Edger, Patrick P.; Puzey, Joshua R.; Kinser, Taliesin; Cheng, Philip; Vernon, Daniel M.; Forsthoefel, Nancy R.; Cooley, Arielle M.

    2017-01-01

    While quantitative PCR (qPCR) is widely recognized as being among the most accurate methods for quantifying gene expression, it is highly dependent on the use of reliable, stably expressed reference genes. With the increased availability of high-throughput methods for measuring gene expression, whole-transcriptome approaches may be increasingly utilized for reference gene selection and validation. In this study, RNA-seq was used to identify a set of novel qPCR reference genes and evaluate a panel of traditional “housekeeping” reference genes in two species of the evolutionary model plant genus Mimulus. More broadly, the methods proposed in this study can be used to harness the power of transcriptomes to identify appropriate reference genes for qPCR in any study organism, including emerging and nonmodel systems. We find that RNA-seq accurately estimates gene expression means in comparison to qPCR, and that expression means are robust to moderate environmental and genetic variation. However, measures of expression variability were only in agreement with qPCR for samples obtained from a shared environment. This result, along with transcriptome-wide comparisons, suggests that environmental changes have greater impacts on expression variability than on expression means. We discuss how this issue can be addressed through experimental design, and suggest that the ever-expanding pool of published transcriptomes represents a rich and low-cost resource for developing better reference genes for qPCR. PMID:28258113

  8. Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards.

    PubMed

    Porter, Joshua R; Telford, William G; Batchelor, Eric

    2017-02-25

    Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

  9. Selection and validation of reference genes for transcript normalization in gene expression studies in Catharanthus roseus.

    PubMed

    Pollier, Jacob; Vanden Bossche, Robin; Rischer, Heiko; Goossens, Alain

    2014-10-01

    Quantitative Real-Time PCR (qPCR), a sensitive and commonly used technique for gene expression analysis, requires stably expressed reference genes for normalization of gene expression. Up to now, only one reference gene for qPCR analysis, corresponding to 40S Ribosomal protein S9 (RPS9), was available for the medicinal plant Catharanthus roseus, the only source of the commercial anticancer drugs vinblastine and vincristine. Here, we screened for additional reference genes for this plant species by mining C. roseus RNA-Seq data for orthologs of 22 genes known to be stably expressed in Arabidopsis thaliana and qualified as superior reference genes for this model plant species. Based on this, eight candidate C. roseus reference genes were identified and, together with RPS9, evaluated by performing qPCR on a series of different C. roseus explants and tissue cultures. NormFinder, geNorm and BestKeeper analyses of the resulting qPCR data revealed that the orthologs of At2g28390 (SAND family protein, SAND), At2g32170 (N2227-like family protein, N2227) and At4g26410 (Expressed protein, EXP) had the highest expression stability across the different C. roseus samples and are superior as reference genes as compared to the traditionally used RPS9. Analysis of publicly available C. roseus RNA-Seq data confirmed the expression stability of SAND and N2227, underscoring their value as reference genes for C. roseus qPCR analysis.

  10. Importance of reference gene selection for articular cartilage mechanobiology studies

    PubMed Central

    Al-Sabah, A.; Stadnik, P.; Gilbert, S.J.; Duance, V.C.; Blain, E.J.

    2016-01-01

    Summary Objective Identification of genes differentially expressed in mechano-biological pathways in articular cartilage provides insight into the molecular mechanisms behind initiation and/or progression of osteoarthritis (OA). Quantitative PCR (qPCR) is commonly used to measure gene expression, and is reliant on the use of reference genes for normalisation. Appropriate validation of reference gene stability is imperative for accurate data analysis and interpretation. This study determined in vitro reference gene stability in articular cartilage explants and primary chondrocytes subjected to different compressive loads and tensile strain, respectively. Design The expression of eight commonly used reference genes (18s, ACTB, GAPDH, HPRT1, PPIA, RPL4, SDHA and YWHAZ) was determined by qPCR and data compared using four software packages (comparative delta-Ct method, geNorm, NormFinder and BestKeeper). Calculation of geometric means of the ranked weightings was carried out using RefFinder. Results Appropriate reference gene(s) for normalisation of mechanically-regulated transcript levels in articular cartilage tissue or isolated chondrocytes were dependent on experimental set-up. SDHA, YWHAZ and RPL4 were the most stable genes whilst glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to a lesser extent Hypoxanthine-guanine phosphoribosyltransferase (HPRT), showed variable expression in response to load, demonstrating their unsuitability in such in vitro studies. The effect of using unstable reference genes to normalise the expression of aggrecan (ACAN) and matrix metalloproteinase 3 (MMP3) resulted in inaccurate quantification of these mechano-sensitive genes and erroneous interpretation/conclusions. Conclusion This study demonstrates that commonly used ‘reference genes’ may be unsuitable for in vitro cartilage chondrocyte mechanobiology studies, reinforcing the principle that careful validation of reference genes is essential prior to each experiment to

  11. Reference genes for normalization of gene expression studies in human osteoarthritic articular cartilage

    PubMed Central

    Pombo-Suarez, Manuel; Calaza, Manuel; Gomez-Reino, Juan J; Gonzalez, Antonio

    2008-01-01

    Background Assessment of gene expression is an important component of osteoarthritis (OA) research, greatly improved by the development of quantitative real-time PCR (qPCR). This technique requires normalization for precise results, yet no suitable reference genes have been identified in human articular cartilage. We have examined ten well-known reference genes to determine the most adequate for this application. Results Analyses of expression stability in cartilage from 10 patients with hip OA, 8 patients with knee OA and 10 controls without OA were done with classical statistical tests and the software programs geNorm and NormFinder. Results from the three methods of analysis were broadly concordant. Some of the commonly used reference genes, GAPDH, ACTB and 18S RNA, performed poorly in our analysis. In contrast, the rarely used TBP, RPL13A and B2M genes were the best. It was necessary to use together several of these three genes to obtain the best results. The specific combination depended, to some extent, on the type of samples being compared. Conclusion Our results provide a satisfactory set of previously unused reference genes for qPCR in hip and knee OA This confirms the need to evaluate the suitability of reference genes in every tissue and experimental situation before starting the quantitative assessment of gene expression by qPCR. PMID:18226276

  12. GETPrime 2.0: gene- and transcript-specific qPCR primers for 13 species including polymorphisms

    PubMed Central

    David, Fabrice P.A.; Rougemont, Jacques; Deplancke, Bart

    2017-01-01

    GETPrime (http://bbcftools.epfl.ch/getprime) is a database with a web frontend providing gene- and transcript-specific, pre-computed qPCR primer pairs. The primers have been optimized for genome-wide specificity and for allowing the selective amplification of one or several splice variants of most known genes. To ease selection, primers have also been ranked according to defined criteria such as genome-wide specificity (with BLAST), amplicon size, and isoform coverage. Here, we report a major upgrade (2.0) of the database: eight new species (yeast, chicken, macaque, chimpanzee, rat, platypus, pufferfish, and Anolis carolinensis) now complement the five already included in the previous version (human, mouse, zebrafish, fly, and worm). Furthermore, the genomic reference has been updated to Ensembl v81 (while keeping earlier versions for backward compatibility) as a result of re-designing the back-end database and automating the import of relevant sections of the Ensembl database in species-independent fashion. This also allowed us to map known polymorphisms to the primers (on average three per primer for human), with the aim of reducing experimental error when targeting specific strains or individuals. Another consequence is that the inclusion of future Ensembl releases and other species has now become a relatively straightforward task. PMID:28053161

  13. Identification of stable reference genes for quantitative PCR in cells derived from chicken lymphoid organs.

    PubMed

    Borowska, D; Rothwell, L; Bailey, R A; Watson, K; Kaiser, P

    2016-02-01

    Quantitative polymerase chain reaction (qPCR) is a powerful technique for quantification of gene expression, especially genes involved in immune responses. Although qPCR is a very efficient and sensitive tool, variations in the enzymatic efficiency, quality of RNA and the presence of inhibitors can lead to errors. Therefore, qPCR needs to be normalised to obtain reliable results and allow comparison. The most common approach is to use reference genes as internal controls in qPCR analyses. In this study, expression of seven genes, including β-actin (ACTB), β-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), TATA box binding protein (TBP), α-tubulin (TUBAT) and 28S ribosomal RNA (r28S), was determined in cells isolated from chicken lymphoid tissues and stimulated with three different mitogens. The stability of the genes was measured using geNorm, NormFinder and BestKeeper software. The results from both geNorm and NormFinder were that the three most stably expressed genes in this panel were TBP, GAPDH and r28S. BestKeeper did not generate clear answers because of the highly heterogeneous sample set. Based on these data we will include TBP in future qPCR normalisation. The study shows the importance of appropriate reference gene normalisation in other tissues before qPCR analysis.

  14. Evaluation of potential reference genes for use in gene expression studies in the conifer pathogen (Heterobasidion annosum).

    PubMed

    Raffaello, Tommaso; Asiegbu, Fred O

    2013-07-01

    The basidiomycete Heterobasidion annosum is the causative agent of butt and root rot disease of conifer trees and it's one of the most destructive conifer pathogen in the northern hemisphere. Because of the intrinsic difficulties in genome manipulation in this fungus, most studies have been focused on gene expression analysis using quantitative real time polymerase chain reaction (qPCR). qPCR is a powerful technique but its reliability resides in the correct selection of a set of reference genes used in the data normalization. In this study, we determined the expression stability of 11 selected reference genes in H. annosum. Almost nothing has so far been published about validation of a set of reference genes to be used in gene expression experiments in this fungus. Eleven reference genes were validated in H. annosum which was grown on three different substrates: pine bark, pine heartwood, and pine sapwood. Bestkeeper and NormFinder Excel-based software were used to evaluate the reference gene transcripts' stability. The results from these two programs indicated that three reference genes namely Tryp metab, RNA Pol3 TF, and Actin were stable in H. annosum in the conditions studied. Interestingly, the GAPDH transcript which has been extensively used in qPCR data normalization is not the best choice when a wide reference gene selection is available. This work represents the first extensive validation of reference genes in H. annosum providing support for gene expression studies and benefits for the wider forest pathology community.

  15. Quantification of Las gene by qPCR from orange juice extracted from Huanglongbing infected fruit

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this research is to establish a methodology to quantify the Candidatus Liberibacter asiaticus (CLas) in orange juice as an indicator of orange juice quality. Current standard method for citrus Huanglongbing (HLB) diagnosis is using real-time Polymerase Chain Reaction (qPCR) to quan...

  16. Superior cross-species reference genes: a blueberry case study.

    PubMed

    Die, Jose V; Rowland, Lisa J

    2013-01-01

    The advent of affordable Next Generation Sequencing technologies has had major impact on studies of many crop species, where access to genomic technologies and genome-scale data sets has been extremely limited until now. The recent development of genomic resources in blueberry will enable the application of high throughput gene expression approaches that should relatively quickly increase our understanding of blueberry physiology. These studies, however, require a highly accurate and robust workflow and make necessary the identification of reference genes with high expression stability for correct target gene normalization. To create a set of superior reference genes for blueberry expression analyses, we mined a publicly available transcriptome data set from blueberry for orthologs to a set of Arabidopsis genes that showed the most stable expression in a developmental series. In total, the expression stability of 13 putative reference genes was evaluated by qPCR and a set of new references with high stability values across a developmental series in fruits and floral buds of blueberry were identified. We also demonstrated the need to use at least two, preferably three, reference genes to avoid inconsistencies in results, even when superior reference genes are used. The new references identified here provide a valuable resource for accurate normalization of gene expression in Vaccinium spp. and may be useful for other members of the Ericaceae family as well.

  17. A validation of 10 feline reference genes for gene expression measurements in snap-frozen tissues.

    PubMed

    Penning, Louis C; Vrieling, Henriette E; Brinkhof, Bas; Riemers, Frank M; Rothuizen, Jan; Rutteman, Gerard R; Hazewinkel, Herman A W

    2007-12-15

    For a proper determination of relative mRNA expression levels with real-time quantitative PCR (Q-PCR) internal standards, such as the expression of reference genes, are of utmost importance. For cats, in contrast to dogs, no validation of reference genes has been published. Our goal was to evaluate frequently used reference genes for the analysis of relative mRNA levels from feline tissues in a SYBR Green-based Q-PCR protocol. First, primers were optimized on mRNA-derived cDNA from liver and kidney tissues of randomly chosen (healthy and diseased) cats. Then, the expression variation and stability of each reference gene within a specific tissue was determined. Dental roots and crowns, heart (left ventricle), renal, liver, lung, and mammary gland tissues from 3 to 11 cats of different breeds, sexes, ages, and disease status were included in this study. Averaging relative stabilities over these six tissues revealed the usefulness of each tested gene as reference gene. In order to compensate for the expression variation of a reference gene within a specific tissue, as much as six reference genes (e.g. RPL17, RPL30, RPS7, YWHAZ, and HPRT) were required to obtain highly reliable data in cat tissues. The optimal set of reference genes depended on the tissue analyzed and should, ideally, be selected and evaluated at the start of each experimental condition. A comparison with a similar evaluation in dogs revealed three issues: (i) most ribosomal genes are suitable in both species; (ii) good non-ribosomal reference genes differ; (iii) more feline than canine reference genes are required for proper analysis.

  18. Evaluation of Reference Genes for Quantitative Real-Time PCR in Songbirds

    PubMed Central

    Zinzow-Kramer, Wendy M.; Horton, Brent M.; Maney, Donna L.

    2014-01-01

    Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbird: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology. PMID:24780145

  19. Apparent Polyploidization after Gamma Irradiation: Pitfalls in the Use of Quantitative Polymerase Chain Reaction (qPCR) for the Estimation of Mitochondrial and Nuclear DNA Gene Copy Numbers

    PubMed Central

    Kam, Winnie W. Y.; Lake, Vanessa; Banos, Connie; Davies, Justin; Banati, Richard

    2013-01-01

    Quantitative polymerase chain reaction (qPCR) has been widely used to quantify changes in gene copy numbers after radiation exposure. Here, we show that gamma irradiation ranging from 10 to 100 Gy of cells and cell-free DNA samples significantly affects the measured qPCR yield, due to radiation-induced fragmentation of the DNA template and, therefore, introduces errors into the estimation of gene copy numbers. The radiation-induced DNA fragmentation and, thus, measured qPCR yield varies with temperature not only in living cells, but also in isolated DNA irradiated under cell-free conditions. In summary, the variability in measured qPCR yield from irradiated samples introduces a significant error into the estimation of both mitochondrial and nuclear gene copy numbers and may give spurious evidence for polyploidization. PMID:23722662

  20. Evaluation and Validation of Reference Genes for Normalization of Quantitative Real-Time PCR Based Gene Expression Studies in Peanut

    PubMed Central

    Cindhuri, Katamreddy Sri; Sharma, Kiran K.

    2013-01-01

    The quantitative real-time PCR (qPCR) based techniques have become essential for gene expression studies and high-throughput molecular characterization of transgenic events. Normalizing to reference gene in relative quantification make results from qPCR more reliable when compared to absolute quantification, but requires robust reference genes. Since, ideal reference gene should be species specific, no single internal control gene is universal for use as a reference gene across various plant developmental stages and diverse growth conditions. Here, we present validation studies of multiple stably expressed reference genes in cultivated peanut with minimal variations in temporal and spatial expression when subjected to various biotic and abiotic stresses. Stability in the expression of eight candidate reference genes including ADH3, ACT11, ATPsyn, CYP2, ELF1B, G6PD, LEC and UBC1 was compared in diverse peanut plant samples. The samples were categorized into distinct experimental sets to check the suitability of candidate genes for accurate and reliable normalization of gene expression using qPCR. Stability in expression of the references genes in eight sets of samples was determined by geNorm and NormFinder methods. While three candidate reference genes including ADH3, G6PD and ELF1B were identified to be stably expressed across experiments, LEC was observed to be the least stable, and hence must be avoided for gene expression studies in peanut. Inclusion of the former two genes gave sufficiently reliable results; nonetheless, the addition of the third reference gene ELF1B may be potentially better in a diverse set of tissue samples of peanut. PMID:24167633

  1. Evaluation and validation of reference genes for normalization of quantitative real-time PCR based gene expression studies in peanut.

    PubMed

    Reddy, Dumbala Srinivas; Bhatnagar-Mathur, Pooja; Cindhuri, Katamreddy Sri; Sharma, Kiran K

    2013-01-01

    The quantitative real-time PCR (qPCR) based techniques have become essential for gene expression studies and high-throughput molecular characterization of transgenic events. Normalizing to reference gene in relative quantification make results from qPCR more reliable when compared to absolute quantification, but requires robust reference genes. Since, ideal reference gene should be species specific, no single internal control gene is universal for use as a reference gene across various plant developmental stages and diverse growth conditions. Here, we present validation studies of multiple stably expressed reference genes in cultivated peanut with minimal variations in temporal and spatial expression when subjected to various biotic and abiotic stresses. Stability in the expression of eight candidate reference genes including ADH3, ACT11, ATPsyn, CYP2, ELF1B, G6PD, LEC and UBC1 was compared in diverse peanut plant samples. The samples were categorized into distinct experimental sets to check the suitability of candidate genes for accurate and reliable normalization of gene expression using qPCR. Stability in expression of the references genes in eight sets of samples was determined by geNorm and NormFinder methods. While three candidate reference genes including ADH3, G6PD and ELF1B were identified to be stably expressed across experiments, LEC was observed to be the least stable, and hence must be avoided for gene expression studies in peanut. Inclusion of the former two genes gave sufficiently reliable results; nonetheless, the addition of the third reference gene ELF1B may be potentially better in a diverse set of tissue samples of peanut.

  2. Reference genes for quantitative PCR in the adipose tissue of mice with metabolic disease.

    PubMed

    Almeida-Oliveira, Fernanda; Leandro, João G B; Ausina, Priscila; Sola-Penna, Mauro; Majerowicz, David

    2017-04-01

    Obesity and diabetes are metabolic diseases and they are increasing in prevalence. The dynamics of gene expression associated with these diseases is fundamental to identifying genes involved in related biological processes. qPCR is a sensitive technique for mRNA quantification and the most commonly used method in gene-expression studies. However, the reliability of these results is directly influenced by data normalization. As reference genes are the major normalization method used, this work aims to identify reference genes for qPCR in adipose tissues of mice with type-I diabetes or obesity. We selected 12 genes that are commonly used as reference genes. The expression of these genes in the adipose tissues of mice was analyzed in the context of three different experimental protocols: 1) untreated animals; 2) high-fat-diet animals; and 3) streptozotocin-treated animals. Gene-expression stability was analyzed using four different algorithms. Our data indicate that TATA-binding protein is stably expressed across adipose tissues in control animals. This gene was also a useful reference when the brown adipose tissues of control and obese mice were analyzed. The mitochondrial ATP synthase F1 complex gene exhibits stable expression in subcutaneous and perigonadal adipose tissue from control and obese mice. Moreover, this gene is the best reference for qPCR normalization in adipose tissue from streptozotocin-treated animals. These results show that there is no perfect stable gene suited for use under all experimental conditions. In conclusion, the selection of appropriate genes is a prerequisite to ensure qPCR reliability and must be performed separately for different experimental protocols.

  3. Detection of stable reference genes for real-time PCR analysis in schizophrenia and bipolar disorder.

    PubMed

    Silberberg, Gilad; Baruch, Kuti; Navon, Ruth

    2009-08-15

    Gene expression studies using postmortem human brain tissue are a common tool for studying the etiology of psychiatric disorders. Quantitative real-time PCR (qPCR) is an accurate and sensitive technique used for gene expression analysis in which the expression level is quantified by normalization to one or more reference genes. Therefore, accurate data normalization is critical for validating results obtained by qPCR. This study aimed to identify genes that may serve as reference in postmortem dorsolateral-prefrontal cortices (Brodmann's area 46) of schizophrenics, bipolar disorder (BPD) patients, and control subjects. In the exploratory stage of the analysis, samples of four BPD patients, two schizophrenics, and two controls were quantified using the TaqMan Low Density Array endogenous control panel, containing assays for 16 commonly used reference genes. In the next stage, six of these genes (TFRC, RPLP0, ACTB, POLR2a, B2M, and GAPDH) were quantified by qPCR in 12 samples of each clinical group. Expressional stability of the genes was determined by GeNorm and NormFinder. TFRC and RPLP0 were the most stably expressed genes, whereas the commonly used 18S, POLR2a, and GAPDH were the least stable. This report stresses the importance of examining expressional stability of candidate reference genes in the specific sample collection to be analyzed.

  4. Selection of suitable reference genes for quantitative gene expression studies in milk somatic cells of lactating cows (Bos indicus).

    PubMed

    Varshney, N; Mohanty, A K; Kumar, S; Kaushik, J K; Dang, A K; Mukesh, M; Mishra, B P; Kataria, R; Kimothi, S P; Mukhopadhyay, T K; Malakar, D; Prakash, B S; Grover, S; Batish, V K

    2012-06-01

    We assessed the suitability of 9 internal control genes (ICG) in milk somatic cells of lactating cows to find suitable reference genes for use in quantitative PCR (qPCR). Eighteen multiparous lactating Sahiwal cows were used, 6 in each of 3 lactation stages: early (25 ± 5 d in milk), mid (160 ± 15 d in milk), and late (275 ± 25 d in milk) lactation. Nine candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), protein phosphatase 1 regulatory subunit 11 (PPP1R11), β-actin (ACTB), β-2 microglobulin (B2M), 40S ribosomal protein S15a (RPS15A), ubiquitously expressed transcript (UXT), mitochondrial GTPase 1 (MTG1), 18S rRNA (RN18S1), and ubiquitin (UBC)] were evaluated. Three genes, β-casein (CSN2), lactoferrin (LTF), and cathelicidin (CAMP) were chosen as target genes. Very high amplification was observed in 7 ICG and very low level amplification was observed in 2 ICG (UXT and MTG1). Thus, UXT and MTG1 were excluded from further analysis. The qPCR data were analyzed by 2 software packages, geNorm and NormFinder, to determine suitable reference genes, based on their stability and expression. Overall, PPP1R11, ACTB, UBC, and GAPDH were stably expressed among all candidate reference genes. Therefore, these genes could be used as ICG for normalization of qPCR data in milk somatic cells through lactation.

  5. Evaluation of reference genes for gene expression studies in human brown adipose tissue.

    PubMed

    Taube, Magdalena; Andersson-Assarsson, Johanna C; Lindberg, Kristin; Pereira, Maria J; Gäbel, Markus; Svensson, Maria K; Eriksson, Jan W; Svensson, Per-Arne

    2015-01-01

    Human brown adipose tissue (BAT) has during the last 5 year been subjected to an increasing research interest, due to its putative function as a target for future obesity treatments. The most commonly used method for molecular studies of human BAT is the quantitative polymerase chain reaction (qPCR). This method requires normalization to a reference gene (genes with uniform expression under different experimental conditions, e.g. similar expression levels between human BAT and WAT), but so far no evaluation of reference genes for human BAT has been performed. Two different microarray datasets with samples containing human BAT were used to search for genes with low variability in expression levels. Seven genes (FAM96B, GNB1, GNB2, HUWE1, PSMB2, RING1 and TPT1) identified by microarray analysis, and 8 commonly used reference genes (18S, B2M, GAPDH, LRP10, PPIA, RPLP0, UBC, and YWHAZ) were selected and further analyzed by quantitative PCR in both BAT containing perirenal adipose tissue and subcutaneous adipose tissue. Results were analyzed using 2 different algorithms (Normfinder and geNorm). Most of the commonly used reference genes displayed acceptably low variability (geNorm M-values <0.5) in the samples analyzed, but the novel reference genes identified by microarray displayed an even lower variability (M-values <0.25). Our data suggests that PSMB2, GNB2 and GNB1 are suitable novel reference genes for qPCR analysis of human BAT and we recommend that they are included in future gene expression studies of human BAT.

  6. Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species.

    PubMed

    Reddy, Dumbala Srinivas; Bhatnagar-Mathur, Pooja; Reddy, Palakolanu Sudhakar; Sri Cindhuri, Katamreddy; Sivaji Ganesh, Adusumalli; Sharma, Kiran Kumar

    2016-01-01

    Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses.

  7. Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species

    PubMed Central

    Reddy, Palakolanu Sudhakar; Sri Cindhuri, Katamreddy; Sivaji Ganesh, Adusumalli; Sharma, Kiran Kumar

    2016-01-01

    Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses. PMID:26863232

  8. qPCR for Second Year Undergraduates: A Short, Structured Inquiry to Illustrate Differential Gene Expression

    ERIC Educational Resources Information Center

    McCauslin, Christine Seitz; Gunn, Kathryn Elaine; Pirone, Dana; Staiger, Jennifer

    2015-01-01

    We describe a structured inquiry laboratory exercise that examines transcriptional regulation of the "NOS2" gene under conditions that simulate the inflammatory response in macrophages. Using quantitative PCR and the comparative C[subscript T] method, students are able determine whether transcriptional activation of "NOS2"…

  9. Normalization of gene expression using SYBR green qPCR: a case for paraoxonase 1 and 2 in Alzheimer's disease brains.

    PubMed

    Leduc, Valérie; Legault, Véronique; Dea, Doris; Poirier, Judes

    2011-08-30

    Validating the expression stability of reference genes is crucial for reliable normalization of real-time quantitative PCR (qPCR) data, but relatively few studies have investigated this issue in brain human tissues. The present study thus aimed at identifying in human post-mortem brain tissues a set of suitable endogenous reference genes (ERG) for the expression analysis of potential candidate genes associated with Alzheimer's disease (AD). The mRNA levels of ten common ERGs (ACTB, GAPDH, GPS1, GUSB, M-RIP, PGK1, POL2RF, PPIA, UBE2D2, and YES1) were determined in the frontal cortex of autopsy-confirmed AD and non-demented control cases (n=20) using SYBR Green technology. Then, these levels were ranked according to their expression stability using three software applications: geNorm, NormFinder and BestKeeper. Whereas PPIA and UBE2D2 were among the ERGs with the most reliable expression, ACTB was the worst. Subsequently, using PPIA and UBE2D2 as ERGs for normalization, the mRNA levels of paraoxonase 1 (PON1) and paraoxonase 2 (PON2) were quantified in the frontal cortex of AD and control cases (n=80) and analyzed using the REST 2009 program. Our results indicate that both paraoxonases are expressed in the human frontal cortex and that PON2 but not PON1 mRNA levels are up-regulated in AD relative to non-demented controls. However, re-analysis of the results by ANCOVA indicated that the significance of the difference between AD and control groups depended upon the ERG used for normalization. The use of a computational method allowing the inclusion of possible confounding factors is thus recommended for the analysis of data.

  10. Nanoliter qPCR platform for highly parallel, quantitative assessment of reductive dehalogenase genes and populations of dehalogenating microorganisms in complex environments.

    PubMed

    Mayer-Blackwell, Koshlan; Azizian, Mohammad F; Machak, Christina; Vitale, Elena; Carpani, Giovanna; de Ferra, Francesca; Semprini, Lewis; Spormann, Alfred M

    2014-08-19

    Idiosyncratic combinations of reductive dehalogenase (rdh) genes are a distinguishing genomic feature of closely related organohalogen-respiring bacteria. This feature can be used to deconvolute the population structure of organohalogen-respiring bacteria in complex environments and to identify relevant subpopulations, which is important for tracking interspecies dynamics needed for successful site remediation. Here we report the development of a nanoliter qPCR platform to identify organohalogen-respiring bacteria and populations by quantifying major orthologous reductive dehalogenase gene groups. The qPCR assays can be operated in parallel within a 5184-well nanoliter qPCR (nL-qPCR) chip at a single annealing temperature and buffer condition. We developed a robust bioinformatics approach to select from thousands of computationally proposed primer pairs those that are specific to individual rdh gene groups and compatible with a single amplification condition. We validated hundreds of the most selective qPCR assays and examined their performance in a trichloroethene-degrading bioreactor, revealing population structures as well as their unexpected shifts in abundance and community dynamics.

  11. Selection of reference genes for gene expression studies in virus-infected monocots using quantitative real-time PCR.

    PubMed

    Zhang, Kun; Niu, Shaofang; Di, Dianping; Shi, Lindan; Liu, Deshui; Cao, Xiuling; Miao, Hongqin; Wang, Xianbing; Han, Chenggui; Yu, Jialin; Li, Dawei; Zhang, Yongliang

    2013-10-10

    Both genome-wide transcriptomic surveys of the mRNA expression profiles and virus-induced gene silencing-based molecular studies of target gene during virus-plant interaction involve the precise estimation of the transcript abundance. Quantitative real-time PCR (qPCR) is the most widely adopted technique for mRNA quantification. In order to obtain reliable quantification of transcripts, identification of the best reference genes forms the basis of the preliminary work. Nevertheless, the stability of internal controls in virus-infected monocots needs to be fully explored. In this work, the suitability of ten housekeeping genes (ACT, EF1α, FBOX, GAPDH, GTPB, PP2A, SAND, TUBβ, UBC18 and UK) for potential use as reference genes in qPCR were investigated in five different monocot plants (Brachypodium, barley, sorghum, wheat and maize) under infection with different viruses including Barley stripe mosaic virus (BSMV), Brome mosaic virus (BMV), Rice black-streaked dwarf virus (RBSDV) and Sugarcane mosaic virus (SCMV). By using three different algorithms, the most appropriate reference genes or their combinations were identified for different experimental sets and their effectiveness for the normalisation of expression studies were further validated by quantitative analysis of a well-studied PR-1 gene. These results facilitate the selection of desirable reference genes for more accurate gene expression studies in virus-infected monocots.

  12. Reference gene for primary culture of prostate cancer cells.

    PubMed

    Souza, Aline Francielle Damo; Brum, Ilma Simoni; Neto, Brasil Silva; Berger, Milton; Branchini, Gisele

    2013-04-01

    Selection of reference genes to normalize mRNA levels between samples is critical for gene expression studies because their expression can vary depending on the tissues or cells used and the experimental conditions. We performed ten cell cultures from samples of prostate cancer. Cells were divided into three groups: control (with no transfection protocol), cells transfected with siRNA specific to knockdown the androgen receptor and cells transfected with inespecific siRNAs. After 24 h, mRNA was extracted and gene expression was analyzed by Real-time qPCR. Nine candidates to reference genes for gene expression studies in this model were analyzed (aminolevulinate, delta-, synthase 1 (ALAS1); beta-actin (ACTB); beta-2-microglobulin (B2M); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine phosphoribosyltransferase 1 (HPRT1); succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA); TATA box binding protein (TBP); ubiquitin C (UBC); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)). Expression stability was calculated NormFinder algorithm to find the most stable genes. NormFinder calculated SDHA as the most stable gene and the gene with the lowest intergroup and intragroup variation, and indicated GAPDH and SDHA as the best combination of two genes for the purpose of normalization. Androgen receptor mRNA expression was evaluated after normalization by each candidate gene and showed statistical difference in the transfected group compared to control group only when normalized by combination of GAPDH and SDHA. Based on the algorithm analysis, the combination of SDHA and GAPDH should be used to normalize target genes mRNA levels in primary culture of prostate cancer cells submitted to transfection with siRNAs.

  13. Evaluation of reference genes at different developmental stages for quantitative real-time PCR in Aedes aegypti.

    PubMed

    Dzaki, Najat; Ramli, Karima N; Azlan, Azali; Ishak, Intan H; Azzam, Ghows

    2017-03-16

    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.

  14. Evaluation of reference genes at different developmental stages for quantitative real-time PCR in Aedes aegypti

    PubMed Central

    Dzaki, Najat; Ramli, Karima N.; Azlan, Azali; Ishak, Intan H.; Azzam, Ghows

    2017-01-01

    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. PMID:28300076

  15. Determination of reference genes for circadian studies in different tissues and mouse strains

    PubMed Central

    2010-01-01

    Background Circadian rhythms have a profound effect on human health. Their disruption can lead to serious pathologies, such as cancer and obesity. Gene expression studies in these pathologies are often studied in different mouse strains by quantitative real time polymerase chain reaction (qPCR). Selection of reference genes is a crucial step of qPCR experiments. Recent studies show that reference gene stability can vary between species and tissues, but none has taken circadian experiments into consideration. Results In the present study the expression of ten candidate reference genes (Actb, Eif2a, Gapdh, Hmbs, Hprt1, Ppib, Rn18s, Rplp0, Tbcc and Utp6c) was measured in 131 liver and 97 adrenal gland samples taken from three mouse strains (C57BL/6JOlaHsd, 129Pas plus C57BL/6J and Crem KO on 129Pas plus C57BL/6J background) every 4 h in a 24 h period. Expression stability was evaluated by geNorm and NormFinder programs. Differences in ranking of the most stable reference genes were observed both between individual mouse strains as well as between tissues within each mouse strain. We show that selection of reference gene (Actb) that is often used for analyses in individual mouse strains leads to errors if used for normalization when different mouse strains are compared. We identified alternative reference genes that are stable in these comparisons. Conclusions Genetic background and circadian time influence the expression stability of reference genes. Differences between mouse strains and tissues should be taken into consideration to avoid false interpretations. We show that the use of a single reference gene can lead to false biological conclusions. This manuscript provides a useful reference point for researchers that search for stable reference genes in the field of circadian biology. PMID:20712867

  16. Reference genes to quantify gene expression during oogenesis in a teleost fish.

    PubMed

    Deloffre, Laurence A M; Andrade, André; Filipe, Alexandra I; Canario, Adelino V M

    2012-09-10

    Understanding the molecular events involved in the acquisition of competence during oogenesis is a key step to determine the secret of 'high quality' eggs for aquaculture. Quantitative real time polymerase chain reaction (qPCR) is the technique of election to determine changes in transcript abundance in such studies, but choosing reference genes for normalization, in particular during oogenesis, remains a challenge. In the present study, transcription of 6 functionally distinct genes, β actin (ACTB), cathepsin D (CTSD), cathepsin Z (CTSZ), elongation factor 1 α (EEF1A), TATA binding protein (TBP) and tubulin A (TUBA1A) was assessed as normalizers of bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI) gene expression in mRNA from Mozambique tilapia oocytes during oogenesis. Reverse transcription was equally efficient and varies little in all samples. Most of the genes considered for reference were stable during early stages of oogenesis but variations were observed during vitellogenesis. A single gene and up to 3 genes were shown to be insufficient for reliable normalization throughout the whole oogenesis. The combination of the genes ACTB, CTSD, EEF1A and CTSZ as reference was found to minimize variation and has the most stable expression pattern between maturation stages.

  17. Reference genes for quantitative gene expression studies in multiple avian species.

    PubMed

    Olias, Philipp; Adam, Iris; Meyer, Anne; Scharff, Constance; Gruber, Achim D

    2014-01-01

    Quantitative real-time PCR (qPCR) rapidly and reliably quantifies gene expression levels across different experimental conditions. Selection of suitable reference genes is essential for meaningful normalization and thus correct interpretation of data. In recent years, an increasing number of avian species other than the chicken has been investigated molecularly, highlighting the need for an experimentally validated pan-avian primer set for reference genes. Here we report testing a set for 14 candidate reference genes (18S, ABL, GAPDH, GUSB, HMBS, HPRT, PGK1, RPL13, RPL19, RPS7, SDHA, TFRC, VIM, YWHAZ) on different tissues of the mallard (Anas platyrhynchos), domestic chicken (Gallus gallus domesticus), common crane (Grus grus), white-tailed eagle (Haliaeetus albicilla), domestic turkey (Meleagris gallopavo f. domestica), cockatiel (Nymphicus hollandicus), Humboldt penguin (Sphenicus humboldti), ostrich (Struthio camelus) and zebra finch (Taeniopygia guttata), spanning a broad range of the phylogenetic tree of birds. Primer pairs for six to 11 genes were successfully established for each of the nine species. As a proof of principle, we analyzed expression levels of 10 candidate reference genes as well as FOXP2 and the immediate early genes, EGR1 and CFOS, known to be rapidly induced by singing in the avian basal ganglia. We extracted RNA from microbiopsies of the striatal song nucleus Area X of adult male zebra finches after they had sang or remained silent. Using three different statistical algorithms, we identified five genes (18S, PGK1, RPS7, TFRC, YWHAZ) that were stably expressed within each group and also between the singing and silent conditions, establishing them as suitable reference genes. In conclusion, the newly developed pan-avian primer set allows accurate normalization and quantification of gene expression levels in multiple avian species.

  18. Validation of reference genes for quantitative real-time PCR during latex regeneration in rubber tree.

    PubMed

    Long, Xiangyu; He, Bin; Gao, Xinsheng; Qin, Yunxia; Yang, Jianghua; Fang, Yongjun; Qi, Jiyan; Tang, Chaorong

    2015-06-01

    In rubber tree, latex regeneration is one of the decisive factors influencing the rubber yield, although its molecular regulation is not well known. Quantitative real-time PCR (qPCR) is a popular and powerful tool used to understand the molecular mechanisms of latex regeneration. However, the suitable reference genes required for qPCR are not available to investigate the expressions of target genes during latex regeneration. In this study, 20 candidate reference genes were selected and evaluated for their expression stability across the samples during the process of latex regeneration. All reference genes showed a relatively wide range of the threshold cycle values, and their stability was validated by four different algorithms (comparative delta Ct method, Bestkeeper, NormFinder and GeNorm). Three softwares (comparative delta Ct method, NormFinder and GeNorm) exported similar results that identify UBC4, ADF, UBC2a, eIF2 and ADF4 as the top five suitable references, and 18S as the least suitable one. The application of the screened references would improve accuracy and reliability of gene expression analysis in latex regeneration experiments.

  19. Selection of Reference Genes for Gene Expression Studies related to lung injury in a preterm lamb model

    PubMed Central

    Pereira-Fantini, Prue M.; Rajapaksa, Anushi E.; Oakley, Regina; Tingay, David G.

    2016-01-01

    Preterm newborns often require invasive support, however even brief periods of supported ventilation applied inappropriately to the lung can cause injury. Real-time quantitative reverse transcriptase-PCR (qPCR) has been extensively employed in studies of ventilation-induced lung injury with the reference gene 18S ribosomal RNA (18S RNA) most commonly employed as the internal control reference gene. Whilst the results of these studies depend on the stability of the reference gene employed, the use of 18S RNA has not been validated. In this study the expression profile of five candidate reference genes (18S RNA, ACTB, GAPDH, TOP1 and RPS29) in two geographical locations, was evaluated by dedicated algorithms, including geNorm, Normfinder, Bestkeeper and ΔCt method and the overall stability of these candidate genes determined (RefFinder). Secondary studies examined the influence of reference gene choice on the relative expression of two well-validated lung injury markers; EGR1 and IL1B. In the setting of the preterm lamb model of lung injury, RPS29 reference gene expression was influenced by tissue location; however we determined that individual ventilation strategies influence reference gene stability. Whilst 18S RNA is the most commonly employed reference gene in preterm lamb lung studies, our results suggest that GAPDH is a more suitable candidate. PMID:27210246

  20. With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies

    PubMed Central

    Chapman, Joanne R.; Waldenström, Jonas

    2015-01-01

    The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies. PMID:26555275

  1. With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies.

    PubMed

    Chapman, Joanne R; Waldenström, Jonas

    2015-01-01

    The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.

  2. Defining suitable reference genes for RT-qPCR analysis on human sertoli cells after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure.

    PubMed

    Ribeiro, Mariana Antunes; dos Reis, Mariana Bisarro; de Moraes, Leonardo Nazário; Briton-Jones, Christine; Rainho, Cláudia Aparecida; Scarano, Wellerson Rodrigo

    2014-11-01

    Quantitative real-time RT-PCR (qPCR) has proven to be a valuable molecular technique to quantify gene expression. There are few studies in the literature that describe suitable reference genes to normalize gene expression data. Studies of transcriptionally disruptive toxins, like tetrachlorodibenzo-p-dioxin (TCDD), require careful consideration of reference genes. The present study was designed to validate potential reference genes in human Sertoli cells after exposure to TCDD. 32 candidate reference genes were analyzed to determine their applicability. geNorm and NormFinder softwares were used to obtain an estimation of the expression stability of the 32 genes and to identify the most suitable genes for qPCR data normalization.

  3. Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane

    PubMed Central

    Xue, Bantong; Guo, Jinlong; Que, Youxiong; Fu, Zhiwei; Wu, Luguang; Xu, Liping

    2014-01-01

    Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential “single copy” genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3—high copy number group, TST-1 and PRR-1—medium copy number group, P4H-1, APRT-2 and CYC-2—low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane. PMID:24857916

  4. Selection of suitable endogenous reference genes for relative copy number detection in sugarcane.

    PubMed

    Xue, Bantong; Guo, Jinlong; Que, Youxiong; Fu, Zhiwei; Wu, Luguang; Xu, Liping

    2014-05-19

    Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential "single copy" genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3--high copy number group, TST-1 and PRR-1--medium copy number group, P4H-1, APRT-2 and CYC-2--low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.

  5. Reference gene selection for quantitative real-time PCR normalization in larvae of three species of Grapholitini (Lepidoptera: Tortricidae).

    PubMed

    Ridgeway, Jaryd A; Timm, Alicia E

    2015-01-01

    Despite the agricultural importance of species in the Grapholitini (Lepidoptera: Tortricidae), and the value of gene expression analysis for improved population management, few gene expression studies based on quantitative real-time PCR (qPCR) have been conducted for this tribe. Part of the reason for this lack of information is that suitable reference genes, which are fundamental for accurate normalization of qPCR studies, have not been identified for the tribe. Thus, the expression stability of six potential reference genes (ACT, AK, COI, EF1, ENO and TUB) was assessed in three different tissues (whole body, midgut and cuticle) of Cryptophlebia peltastica (Meyrick), Cydia pomonella (L.) and Thaumatotibia leucotreta (Meyrick). Additionally, these reference genes were tested using T. leucotreta at different temperatures (15°C, 25°C and 35°C) with and without baculovirus infection. Suitable reference genes were identified for the whole body and midgut tissue of all three species, and for cuticle tissue of Cy. pomonella and T. leucotreta. When T. leucotreta was infected with the virus at all temperature conditions ACT, AK and EF1 were found to be the most suitable reference genes for experimental normalization. In general, for all tissue types, species and stress conditions, AK and EF1 were the best-performing reference genes. However, even though the three species analysed were closely related and within the same tribe, each species required varying gene combinations for suitable normalization. This study provides the first reference gene evaluation for the Tortricidae, and paves the way for future qPCR analysis in Tortricidae.

  6. Suitable Reference Genes for Accurate Gene Expression Analysis in Parsley (Petroselinum crispum) for Abiotic Stresses and Hormone Stimuli

    PubMed Central

    Li, Meng-Yao; Song, Xiong; Wang, Feng; Xiong, Ai-Sheng

    2016-01-01

    Parsley, one of the most important vegetables in the Apiaceae family, is widely used in the food, medicinal, and cosmetic industries. Recent studies on parsley mainly focus on its chemical composition, and further research involving the analysis of the plant's gene functions and expressions is required. qPCR is a powerful method for detecting very low quantities of target transcript levels and is widely used to study gene expression. To ensure the accuracy of results, a suitable reference gene is necessary for expression normalization. In this study, four software, namely geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the expression stabilities of eight candidate reference genes of parsley (GAPDH, ACTIN, eIF-4α, SAND, UBC, TIP41, EF-1α, and TUB) under various conditions, including abiotic stresses (heat, cold, salt, and drought) and hormone stimuli treatments (GA, SA, MeJA, and ABA). Results showed that EF-1α and TUB were the most stable genes for abiotic stresses, whereas EF-1α, GAPDH, and TUB were the top three choices for hormone stimuli treatments. Moreover, EF-1α and TUB were the most stable reference genes among all tested samples, and UBC was the least stable one. Expression analysis of PcDREB1 and PcDREB2 further verified that the selected stable reference genes were suitable for gene expression normalization. This study can guide the selection of suitable reference genes in gene expression in parsley. PMID:27746803

  7. Suitable Reference Genes for Accurate Gene Expression Analysis in Parsley (Petroselinum crispum) for Abiotic Stresses and Hormone Stimuli.

    PubMed

    Li, Meng-Yao; Song, Xiong; Wang, Feng; Xiong, Ai-Sheng

    2016-01-01

    Parsley, one of the most important vegetables in the Apiaceae family, is widely used in the food, medicinal, and cosmetic industries. Recent studies on parsley mainly focus on its chemical composition, and further research involving the analysis of the plant's gene functions and expressions is required. qPCR is a powerful method for detecting very low quantities of target transcript levels and is widely used to study gene expression. To ensure the accuracy of results, a suitable reference gene is necessary for expression normalization. In this study, four software, namely geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the expression stabilities of eight candidate reference genes of parsley (GAPDH, ACTIN, eIF-4α, SAND, UBC, TIP41, EF-1α, and TUB) under various conditions, including abiotic stresses (heat, cold, salt, and drought) and hormone stimuli treatments (GA, SA, MeJA, and ABA). Results showed that EF-1α and TUB were the most stable genes for abiotic stresses, whereas EF-1α, GAPDH, and TUB were the top three choices for hormone stimuli treatments. Moreover, EF-1α and TUB were the most stable reference genes among all tested samples, and UBC was the least stable one. Expression analysis of PcDREB1 and PcDREB2 further verified that the selected stable reference genes were suitable for gene expression normalization. This study can guide the selection of suitable reference genes in gene expression in parsley.

  8. Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis.

    PubMed

    dos Santos, Odelta; de Vargas Rigo, Graziela; Frasson, Amanda Piccoli; Macedo, Alexandre José; Tasca, Tiana

    2015-01-01

    Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis.

  9. Appropriate Real-Time PCR Reference Genes for Fluoride Treatment Studies Performed In Vitro or In Vivo

    PubMed Central

    Faibish, D.; Suzuki, M.; Bartlett, J.D.

    2015-01-01

    Objective Quantitative real-time PCR (qPCR) is routinely performed for experiments designed to identify the molecular mechanisms involved in the pathogenesis of dental fluorosis. Expression of reference gene(s) is expected to remain unchanged in fluoride-treated cells or in rodents relative to the corresponding untreated controls. The aim of this study was to select optimal reference genes for fluoride experiments performed in vitro and in vivo. Design Five candidate genes were evaluated: B2m, Eef1a1, Gapdh, Hprt and Tbp. For in vitro experiments, LS8 cells derived from mouse enamel organ were treated with 0, 1, 3 and/or 5 mM sodium fluoride (NaF) for 6 or 18 hours followed by RNA isolation. For in vivo experiments, six-week old rats were treated with 0 or 100 ppm fluoride as NaF for six weeks at which time RNA was isolated from enamel organs. RNA from cells and enamel organs were reverse-transcribed and stability of gene expression for the candidate reference genes was evaluated by qPCR in treated versus non-treated samples. Results The most stably expressed genes in vitro according to geNorm were B2m and Tbp, and according to Normfinder were Hprt and Gapdh. The most stable genes in vivo were Eef1a1 and Gapdh. Expression of Ddit3, a gene previously shown to be induced by fluoride, was demonstrated to be accurately calculated only when using an optimal reference gene. Conclusions This study identifies suitable reference genes for relative quantification of gene expression by qPCR after fluoride treatment both in cultured cells and in the rodent enamel organ. PMID:26615575

  10. Identification of reference genes for circulating microRNA analysis in colorectal cancer

    PubMed Central

    Niu, Yanqin; Wu, Yike; Huang, Jinyong; Li, Qing; Kang, Kang; Qu, Junle; Li, Furong; Gou, Deming

    2016-01-01

    Quantitative real-time PCR (qPCR) is the most frequently used method for measuring expression levels of microRNAs (miRNAs), which is based on normalization to endogenous references. Although circulating miRNAs have been regarded as potential non-invasive biomarker of disease, no study has been performed so far on reference miRNAs for normalization in colorectal cancer. In this study we tried to identify optimal reference miRNAs for qPCR analysis across colorectal cancer patients and healthy individuals. 485 blood-derived miRNAs were profiled in serum sample pools of both colorectal cancer and healthy control. Seven candidate miRNAs chosen from profiling results as well as three previous reported reference miRNAs were validated using qPCR in 30 colorectal cancer patients and 30 healthy individuals, and thereafter analyzed by statistical algorithms BestKeeper, geNorm and NormFinder. Taken together, hsa-miR-93-5p, hsa-miR-25-3p and hsa-miR-106b-5p were recommended as a set of suitable reference genes. More interestingly, the three miRNAs validated from 485 miRNAs are derived from a single primary transcript, indicting the cluster may be highly conserved in colorectal cancer. However, all three miRNAs differed significantly between healthy individuals and non-small cell lung cancer or breast cancer patients and could not be used as reference genes in the two types of cancer. PMID:27759076

  11. Reference gene selection for reverse transcription quantitative polymerase chain reaction in chicken hypothalamus under different feeding status.

    PubMed

    Simon, Á; Jávor, A; Bai, P; Oláh, J; Czeglédi, L

    2017-03-15

    This study was designed to investigate the stability of 10 candidate reference genes, namely ACTB, B2M, GAPDH, HMBS, LBR, POLR2B, RN18S, RPS17, TBP, and YWHAZ for the normalization of gene expression data obtained by quantitative real-time polymerase chain reaction (qPCR) in studies related to feed intake of chicken. Samples were isolated from hypothalamus under three different nutritional status (ad libitum, fasted for 24 hr, fasted for 24 hr then refed for 2 hr). Five different algorithms were applied for the analysis of reference gene stability: BestKeeper, geNorm, NormFinder, the comparative ΔCt method, and a novel approach using multivariate linear mixed-effects modelling for stable reference gene selection. TBP and POLR2B were identified as the two most suitable and B2M and RN18S as the two least stable reference genes for normalization. Despite our review, the current literature showing that RN18S is one of the most commonly used reference gene in chicken gene expression studies, its applicability for normalization should be evaluated before each qPCR experiment.

  12. Validation of Reference Genes for Oral Cancer Detection Panels in a Prospective Blinded Cohort

    PubMed Central

    Martin, Jack L.

    2016-01-01

    Background Reference genes are needed as internal controls to determine relative expression for clinical application of gene expression panels. Candidate constitutively expressed genes must be validated as suitable reference genes in each body fluid and disease entity. Prior studies have predominantly validated oral squamous cell carcinoma associated messenger RNAs (mRNAs) based on quantitative polymerase chain reaction (qPCR) quantification cycle (Cq) values without adjustment for housekeeping genes. Methods One hundred sixty eight patients had saliva collected before clinically driven biopsy of oral lesions suspicious for cancer. Seven potential housekeeping mRNAs and six pre-specified oral cancer associated mRNAs were measured with qPCR by personnel blinded to tissue diagnosis. Housekeeping gene stability was determined with the NormFinder program in a training set of 12 randomly selected cancer and 24 control patients. Genes with stability indices <0.02 were then tested in the validation set consisting of the remaining cancer and control patients and were further validated by the geNorm program. Cancer gene delta Cqs were compared in case and control patients after subtracting the geometric mean of the reference gene raw Cqs. Results B2M and UBC had stability indices >0.02 in the training set and were not further tested. MT-ATP6, RPL30, RPL37A, RPLP0 and RPS17 all had stability indices <0.02 in the training set and in the verification set. The geNorm M values were all ≤1.10. All six pre-specified cancer genes (IL8, IL1, SAT, OAZ1, DUSP1 and S100P) were up-regulated in cancer versus control patients with from nearly twofold to over threefold higher levels (p<0.01 for all based on delta Cq values). Conclusions Five reference genes are validated for use in oral cancer salivary gene expression panels. Six pre-specified oral carcinoma associated genes are demonstrated to be highly significantly up-regulated in cancer patients based on delta Cq values. These cancer

  13. Selection of reference genes for analysis of stress-responsive genes after challenge with viruses and temperature changes in the silkworm Bombyx mori.

    PubMed

    Guo, Huizhen; Jiang, Liang; Xia, Qingyou

    2016-04-01

    Viruses and high temperature (HT) are the primary threats to silkworms. Changes in the expression of stress-response genes can be measured using quantitative polymerase chain reaction (qPCR) after exposure to viruses or HT. However, appropriate reference genes (RGs) for qPCR data normalization have not been established in this organism. In this study, we summarized the RGs used in the previous silkworm studies after infection with Bombyx mori nucleopolyhedrovirus (BmNPV), B. mori cytoplasmic polyhedrosis virus (BmCPV), or B. mori densovirus (BmDNV) or after HT treatment. The expression levels of these RGs were extracted from silkworm transcriptome data to screen for candidate RGs that were unaffected by the experimental conditions. Actin-1 (A1), actin-3 (A3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation initiation factor 4a (TIF-4A) were selected for further qPCR verification. The results of RNA-seq and qPCR showed that GAPDH and TIF-4A were suitable RGs after BmNPV challenge or HT stress, whereas TIF-4A was an appropriate RG for BmCPV or BmDNV-Z challenge in silkworms. These results suggested that TIF-4A may be the most appropriate RG for gene expression analysis after challenge with viruses or HT in silkworms.

  14. TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR.

    PubMed

    Zhang, Qian; Wang, Jing; Deng, Fang; Yan, Zhengjian; Xia, Yinglin; Wang, Zhongliang; Ye, Jixing; Deng, Youlin; Zhang, Zhonglin; Qiao, Min; Li, Ruifang; Denduluri, Sahitya K; Wei, Qiang; Zhao, Lianggong; Lu, Shun; Wang, Xin; Tang, Shengli; Liu, Hao; Luu, Hue H; Haydon, Rex C; He, Tong-Chuan; Jiang, Li

    2015-01-01

    The advent of fluorescence-based quantitative real-time PCR (qPCR) has revolutionized the quantification of gene expression analysis in many fields, including life sciences, agriculture, forensic science, molecular diagnostics, and medicine. While SYBR Green-based qPCR is the most commonly-used platform due to its inexpensive nature and robust chemistry, quantifying the expression of genes with low abundance or RNA samples extracted from highly restricted or limited sources can be challenging because the detection sensitivity of SYBR Green-based qPCR is limited. Here, we develop a novel and effective touchdown qPCR (TqPCR) protocol by incorporating a 4-cycle touchdown stage prior to the quantification amplification stage. Using the same cDNA templates, we find that TqPCR can reduce the average Cq values for Gapdh, Rps13, and Hprt1 reference genes by 4.45, 5.47, and 4.94 cycles, respectively, when compared with conventional qPCR; the overall average Cq value reduction for the three reference genes together is 4.95. We further find that TqPCR can improve PCR amplification efficiency and thus increase detection sensitivity. When the quantification of Wnt3A-induced target gene expression in mesenchymal stem cells is analyzed, we find that, while both conventional qPCR and TqPCR can detect the up-regulation of the relatively abundant target Axin2, only TqPCR can detect the up-regulation of the lowly-expressed targets Oct4 and Gbx2. Finally, we demonstrate that the MRQ2 and MRQ3 primer pairs derived from mouse reference gene Tbp can be used to validate the RNA/cDNA integrity of qPCR samples. Taken together, our results strongly suggest that TqPCR may increase detection sensitivity and PCR amplification efficiency. Overall, TqPCR should be advantageous over conventional qPCR in expression quantification, especially when the transcripts of interest are lowly expressed, and/or the availability of total RNA is highly restricted or limited.

  15. TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR

    PubMed Central

    Zhang, Qian; Wang, Jing; Deng, Fang; Yan, Zhengjian; Xia, Yinglin; Wang, Zhongliang; Ye, Jixing; Deng, Youlin; Zhang, Zhonglin; Qiao, Min; Li, Ruifang; Denduluri, Sahitya K.; Wei, Qiang; Zhao, Lianggong; Lu, Shun; Wang, Xin; Tang, Shengli; Liu, Hao; Luu, Hue H.; Haydon, Rex C.; He, Tong-Chuan; Jiang, Li

    2015-01-01

    The advent of fluorescence-based quantitative real-time PCR (qPCR) has revolutionized the quantification of gene expression analysis in many fields, including life sciences, agriculture, forensic science, molecular diagnostics, and medicine. While SYBR Green-based qPCR is the most commonly-used platform due to its inexpensive nature and robust chemistry, quantifying the expression of genes with low abundance or RNA samples extracted from highly restricted or limited sources can be challenging because the detection sensitivity of SYBR Green-based qPCR is limited. Here, we develop a novel and effective touchdown qPCR (TqPCR) protocol by incorporating a 4-cycle touchdown stage prior to the quantification amplification stage. Using the same cDNA templates, we find that TqPCR can reduce the average Cq values for Gapdh, Rps13, and Hprt1 reference genes by 4.45, 5.47, and 4.94 cycles, respectively, when compared with conventional qPCR; the overall average Cq value reduction for the three reference genes together is 4.95. We further find that TqPCR can improve PCR amplification efficiency and thus increase detection sensitivity. When the quantification of Wnt3A-induced target gene expression in mesenchymal stem cells is analyzed, we find that, while both conventional qPCR and TqPCR can detect the up-regulation of the relatively abundant target Axin2, only TqPCR can detect the up-regulation of the lowly-expressed targets Oct4 and Gbx2. Finally, we demonstrate that the MRQ2 and MRQ3 primer pairs derived from mouse reference gene Tbp can be used to validate the RNA/cDNA integrity of qPCR samples. Taken together, our results strongly suggest that TqPCR may increase detection sensitivity and PCR amplification efficiency. Overall, TqPCR should be advantageous over conventional qPCR in expression quantification, especially when the transcripts of interest are lowly expressed, and/or the availability of total RNA is highly restricted or limited. PMID:26172450

  16. Identification of appropriate reference genes for human mesenchymal stem cell analysis by quantitative real-time PCR.

    PubMed

    Li, Xiuying; Yang, Qiwei; Bai, Jinping; Xuan, Yali; Wang, Yimin

    2015-01-01

    Normalization to a reference gene is the method of choice for quantitative reverse transcription-PCR (RT-qPCR) analysis. The stability of reference genes is critical for accurate experimental results and conclusions. We have evaluated the expression stability of eight commonly used reference genes found in four different human mesenchymal stem cells (MSC). Using geNorm, NormFinder and BestKeeper algorithms, we show that beta-2-microglobulin and peptidyl-prolylisomerase A were the optimal reference genes for normalizing RT-qPCR data obtained from MSC, whereas the TATA box binding protein was not suitable due to its extensive variability in expression. Our findings emphasize the significance of validating reference genes for qPCR analyses. We offer a short list of reference genes to use for normalization and recommend some commercially-available software programs as a rapid approach to validate reference genes. We also demonstrate that the two reference genes, β-actin and glyceraldehyde-3-phosphate dehydrogenase, are frequently used are not always successful in many cases.

  17. Verification of suitable and reliable reference genes for quantitative real-time PCR during adipogenic differentiation in porcine intramuscular stromal-vascular cells.

    PubMed

    Li, X; Huang, K; Chen, F; Li, W; Sun, S; Shi, X-E; Yang, G

    2016-06-01

    Intramuscular fat (IMF) is an important trait influencing meat quality, and intramuscular stromal-vascular cell (MSVC) differentiation is a key factor affecting IMF deposition. Quantitative real-time PCR (qPCR) is often used to screen the differentially expressed genes during differentiation of MSVCs, where proper reference genes are essential. In this study, we assessed 31 of previously reported reference genes for their expression suitability in porcine MSVCs derived form longissimus dorsi with qPCR. The expression stability of these genes was evaluated using NormFinder, geNorm and BestKeeper algorithms. NormFinder and geNorm uncovered ACTB, ALDOA and RPS18 as the most three stable genes. BestKeeper identified RPL13A, SSU72 and DAK as the most three stable genes. GAPDH was found to be the least stable gene by all of the three software packages, indicating it is not an appropriate reference gene in qPCR assay. These results might be helpful for further studies in pigs that explore the molecular mechanism underlying IMF deposition.

  18. QPCR: Application for real-time PCR data management and analysis

    PubMed Central

    Pabinger, Stephan; Thallinger, Gerhard G; Snajder, René; Eichhorn, Heiko; Rader, Robert; Trajanoski, Zlatko

    2009-01-01

    Background Since its introduction quantitative real-time polymerase chain reaction (qPCR) has become the standard method for quantification of gene expression. Its high sensitivity, large dynamic range, and accuracy led to the development of numerous applications with an increasing number of samples to be analyzed. Data analysis consists of a number of steps, which have to be carried out in several different applications. Currently, no single tool is available which incorporates storage, management, and multiple methods covering the complete analysis pipeline. Results QPCR is a versatile web-based Java application that allows to store, manage, and analyze data from relative quantification qPCR experiments. It comprises a parser to import generated data from qPCR instruments and includes a variety of analysis methods to calculate cycle-threshold and amplification efficiency values. The analysis pipeline includes technical and biological replicate handling, incorporation of sample or gene specific efficiency, normalization using single or multiple reference genes, inter-run calibration, and fold change calculation. Moreover, the application supports assessment of error propagation throughout all analysis steps and allows conducting statistical tests on biological replicates. Results can be visualized in customizable charts and exported for further investigation. Conclusion We have developed a web-based system designed to enhance and facilitate the analysis of qPCR experiments. It covers the complete analysis workflow combining parsing, analysis, and generation of charts into one single application. The system is freely available at PMID:19712446

  19. Identification of Appropriate Reference Genes for qRT-PCR Analysis of Heat-Stressed Mammary Epithelial Cells in Riverine Buffaloes (Bubalus bubalis).

    PubMed

    Kapila, Neha; Kishore, Amit; Sodhi, Monika; Sharma, Ankita; Kumar, Pawan; Mohanty, A K; Jerath, Tanushri; Mukesh, M

    2013-01-01

    Gene expression studies require appropriate normalization methods for proper evaluation of reference genes. To date, not many studies have been reported on the identification of suitable reference genes in buffaloes. The present study was undertaken to determine the panel of suitable reference genes in heat-stressed buffalo mammary epithelial cells (MECs). Briefly, MEC culture from buffalo mammary gland was exposed to 42 °C for one hour and subsequently allowed to recover at 37 °C for different time intervals (from 30 m to 48 h). Three different algorithms, geNorm, NormFinder, and BestKeeper softwares, were used to evaluate the stability of 16 potential reference genes from different functional classes. Our data identified RPL4, EEF1A1, and RPS23 genes to be the most appropriate reference genes that could be utilized for normalization of qPCR data in heat-stressed buffalo MECs.

  20. Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages.

    PubMed

    Espínola, Sergio Martin; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

    2014-01-01

    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

  1. Selection and Validation of Reference Genes for Quantitative Real-time PCR in Gentiana macrophylla

    PubMed Central

    He, Yihan; Yan, Hailing; Hua, Wenping; Huang, Yaya; Wang, Zhezhi

    2016-01-01

    Real time quantitative PCR (RT-qPCR or qPCR) has been extensively applied for analyzing gene expression because of its accuracy, sensitivity, and high throughput. However, the unsuitable choice of reference gene(s) can lead to a misinterpretation of results. We evaluated the stability of 10 candidates – five traditional housekeeping genes (UBC21, GAPC2, EF-1α4, UBQ10, and UBC10) and five novel genes (SAND1, FBOX, PTB1, ARP, and Expressed1) – using the transcriptome data of Gentiana macrophylla. Common statistical algorithms ΔCt, GeNorm, NormFinder, and BestKeeper were run with samples collected from plants under various experimental conditions. For normalizing expression levels from tissues at different developmental stages, GAPC2 and UBC21 had the highest rankings. Both SAND1 and GAPC2 proved to be the optimal reference genes for roots from plants exposed to abiotic stresses while EF-1α4 and SAND1 were optimal when examining expression data from the leaves of stressed plants. Based on a comprehensive ranking of stability under different experimental conditions, we recommend that SAND1 and EF-1α4 are the most suitable overall. In this study, to find a suitable reference gene and its real-time PCR assay for G. macrophylla DNA content quantification, we evaluated three target genes including WRKY30, G10H, and SLS, through qualitative and absolute quantitative PCR with leaves under elicitors stressed experimental conditions. Arbitrary use of reference genes without previous evaluation can lead to a misinterpretation of the data. Our results will benefit future research on the expression of genes related to secoiridoid biosynthesis in this species under different experimental conditions. PMID:27446172

  2. Selection of Reference Genes for Expression Study in Pulp and Seeds of Theobroma grandiflorum (Willd. ex Spreng.) Schum

    PubMed Central

    Ferraz dos Santos, Lucas; Santana Silva, Raner José; Oliveira Jordão do Amaral, Daniel; Barbosa de Paula, Márcia Fabiana; Ludke Falcão, Loeni; Legavre, Thierry; Alves, Rafael Moyses

    2016-01-01

    Cupuassu (Theobroma grandiflorum [Willd. ex Spreng.] Schum) is a species of high economic importance in Brazil with great potential at international level due to the multiple uses of both its seeds and pulp in the industry of sweets and cosmetics. For this reason, the cupuassu breeding program focused on the selection of genotypes with high pulp and seed quality—selection associated with the understanding of the mechanisms involved in fruit formation. Gene expression is one of the most used approaches related to such understanding. In this sense, quantitative real-time PCR (qPCR) is a powerful tool, since it rapidly and reliably quantifies gene expression levels across different experimental conditions. The analysis by qPCR and the correct interpretation of data depend on signal normalization using reference genes, i.e. genes presenting a uniform pattern of expression in the analyzed samples. Here, we selected and analyzed the expression of five genes from cupuassu (ACP, ACT, GAPDH, MDH, TUB) to be used as candidates for reference genes on pulp and seed of young, maturing and mature cupuassu fruits. The evaluation of the gene expression stability was obtained using the NormFinder, geNorm and BestKeeper programs. In general, our results indicated that the GAPDH and MDH genes constituted the best combination as reference genes to analyze the expression of cupuassu samples. To our knowledge, this is the first report of reference gene definition in cupuassu, and these results will support subsequent analysis related to gene expression studies in cupuassu plants subjected to different biotic or abiotic conditions as well as serve as a tool for diversity analysis based on pulp and seed quality. PMID:27501324

  3. Selection of Reference Genes for Expression Study in Pulp and Seeds of Theobroma grandiflorum (Willd. ex Spreng.) Schum.

    PubMed

    Ferraz Dos Santos, Lucas; Santana Silva, Raner José; Oliveira Jordão do Amaral, Daniel; Barbosa de Paula, Márcia Fabiana; Ludke Falcão, Loeni; Legavre, Thierry; Alves, Rafael Moyses; Marcellino, Lucilia Helena; Micheli, Fabienne

    2016-01-01

    Cupuassu (Theobroma grandiflorum [Willd. ex Spreng.] Schum) is a species of high economic importance in Brazil with great potential at international level due to the multiple uses of both its seeds and pulp in the industry of sweets and cosmetics. For this reason, the cupuassu breeding program focused on the selection of genotypes with high pulp and seed quality-selection associated with the understanding of the mechanisms involved in fruit formation. Gene expression is one of the most used approaches related to such understanding. In this sense, quantitative real-time PCR (qPCR) is a powerful tool, since it rapidly and reliably quantifies gene expression levels across different experimental conditions. The analysis by qPCR and the correct interpretation of data depend on signal normalization using reference genes, i.e. genes presenting a uniform pattern of expression in the analyzed samples. Here, we selected and analyzed the expression of five genes from cupuassu (ACP, ACT, GAPDH, MDH, TUB) to be used as candidates for reference genes on pulp and seed of young, maturing and mature cupuassu fruits. The evaluation of the gene expression stability was obtained using the NormFinder, geNorm and BestKeeper programs. In general, our results indicated that the GAPDH and MDH genes constituted the best combination as reference genes to analyze the expression of cupuassu samples. To our knowledge, this is the first report of reference gene definition in cupuassu, and these results will support subsequent analysis related to gene expression studies in cupuassu plants subjected to different biotic or abiotic conditions as well as serve as a tool for diversity analysis based on pulp and seed quality.

  4. A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR

    PubMed Central

    Maier, Helena J.; Van Borm, Steven; Young, John R.; Fife, Mark

    2016-01-01

    Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes, selected for the lowest variation in representative samples, can be used to control random technical variation. Reference gene assays must be reliable, have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios: a tissue collection and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where its use might provide misleading results. Methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology. PMID:27537060

  5. Identification of pBC218/pBC210 Genes of Bacillus cereus G9241 in Five Florida Soils Using qPCR.

    PubMed

    Luna, Vicki Ann; Nguyen, Kimmy; Gilling, Damian H

    2014-01-01

    The distribution of the virulent plasmid pBC210 of B. cereus that carries several B. anthracis genes and has been implicated in lethal anthrax-like pulmonary disease is unknown. We screened our collection of 103 B. cereus isolates and 256 soil samples using a quantitative PCR (qPCR) assay that targeted three open reading frames putatively unique to pBC210. When tested with DNA from 2 B. cereus strains carrying pBC210, and 64 Gram-positive and 55 Gram-negative bacterial species, the assay had 100% sensitivity and specificity. None of the DNA from the B. cereus isolates yielded positive amplicons but DNA extracted from five soils collected in Florida gave positive results for all three target sequences of pBC210. While screening confirms that pBC210 is uncommon in B. cereus, this study is the first to report that pBC210 is present in Florida soils. This study improves our knowledge of the distribution of pBC210 in soils and, of public health importance, the potential threat of B. cereus isolates carrying the toxin-carrying plasmid. We demonstrated that sequences of pBC210 can be found in a larger geographical area than previously thought and that finding more B. cereus carrying the virulent plasmid is a possibility in the future.

  6. Selection and evaluation of reference genes for improved interrogation of microbial transcriptomes: case study with the extremophile Acidithiobacillus ferrooxidans

    PubMed Central

    Nieto, Pamela A; Covarrubias, Paulo C; Jedlicki, Eugenia; Holmes, David S; Quatrini, Raquel

    2009-01-01

    Background Normalization is a prerequisite for accurate real time PCR (qPCR) expression analysis and for the validation of microarray profiling data in microbial systems. The choice and use of reference genes that are stably expressed across samples, experimental conditions and designs is a key consideration for the accurate interpretation of gene expression data. Results Here, we evaluate a carefully selected set of reference genes derived from previous microarray-based transcriptional profiling experiments performed on Acidithiobacillus ferrooxidans and identify a set of genes with minimal variability under five different experimental conditions that are frequently used in Acidithiobacilli research. Suitability of these and other previously reported reference genes to monitor the expression of four selected target genes from A. ferrooxidans grown with different energy sources was investigated. Utilization of reference genes map, rpoC, alaS and era results in improved interpretation of gene expression profiles in A. ferrooxidans. Conclusion This investigation provides a validated set of reference genes for studying A. ferrooxidans gene expression under typical biological conditions and an initial point of departure for exploring new experimental setups in this microorganism and eventually in other closely related Acidithiobacilli. The information could also be of value for future transcriptomic experiments in other bacterial systems. PMID:19555508

  7. Identification of suitable reference gene and biomarkers of serum miRNAs for osteoporosis

    PubMed Central

    Chen, Jian; Li, Kai; Pang, Qianqian; Yang, Chao; Zhang, Hongyu; Wu, Feng; Cao, Hongqing; Liu, Hongju; Wan, Yumin; Xia, Weibo; Wang, Jinfu; Dai, Zhongquan; Li, Yinghui

    2016-01-01

    Our objective was to identify suitable reference genes in serum miRNA for normalization and screen potential new biomarkers for osteoporosis diagnosis by a systematic study. Two types of osteoporosis models were used like as mechanical unloading and estrogen deficiency. Through a large-scale screening using microarray, qPCR validation and statistical algorithms, we first identified miR-25-3p as a suitable reference gene for both type of osteoporosis, which also showed stability during the differentiation processes of osteoblast and osteoclast. Then 15 serum miRNAs with differential expression in OVX rats were identified by microarray and qPCR validation. We further detected these 15 miRNAs in postmenopausal women and bedrest rhesus monkeys and evaluated their diagnostic value by ROC analysis. Among these miRNAs, miR-30b-5p was significantly down-regulated in postmenopausal women with osteopenia or osteoporosis; miR-103-3p, miR-142-3p, miR-328-3p were only significantly decreased in osteoporosis. They all showed positive correlations with BMD. Except miR328-3p, the other three miRNAs were also declined in the rhesus monkeys after long-duration bedrest. Their AUC values (all >0.75) proved the diagnostic potential. Our results provided a reliable normalization reference gene and verified a group of circulating miRNAs as non-invasive biomarkers in the detection of postmenopausal- and mechanical unloading- osteoporosis. PMID:27821865

  8. Identification of reference genes for quantitative expression analysis of microRNAs and mRNAs in barley under various stress conditions.

    PubMed

    Ferdous, Jannatul; Li, Yuan; Reid, Nicolas; Langridge, Peter; Shi, Bu-Jun; Tricker, Penny J

    2015-01-01

    For accurate and reliable gene expression analysis using quantitative real-time reverse transcription PCR (qPCR), the selection of appropriate reference genes as an internal control for normalization is crucial. We hypothesized that non-coding, small nucleolar RNAs (snoRNAs)would be stably expressed in different barley varieties and under different experimental treatments,in different tissues and at different developmental stages of plant growth and therefore might prove to be suitable reference genes for expression analysis of both microRNAs (miRNAs)and mRNAs. In this study, we examined the expression stability of ten candidate reference genes in six barley genotypes under five experimental stresses, drought, fungal infection,boron toxicity, nutrient deficiency and salinity. We compared four commonly used housekeeping genes; Actin (ACT), alpha-Tubulin (α-TUB), Glycolytic glyceraldehyde-3-phosphate dehydrogenase(GAPDH), ADP-ribosylation factor 1-like protein (ADP), four snoRNAs; (U18,U61, snoR14 and snoR23) and two microRNAs (miR168, miR159) as candidate reference genes. We found that ADP, snoR14 and snoR23 were ranked as the best of these candidates across diverse samples. Additionally, we found that miR168 was a suitable reference gene for expression analysis in barley. Finally, we validated the performance of our stable and unstable candidate reference genes for both mRNA and miRNA qPCR data normalization under different stress conditions and demonstrated the superiority of the stable candidates. Our data demonstrate the suitability of barley snoRNAs and miRNAs as potential reference genes form iRNA and mRNA qPCR data normalization under different stress treatments [corrected].

  9. Reference gene selection for quantitative real-time PCR normalization in Caragana intermedia under different abiotic stress conditions.

    PubMed

    Zhu, Jianfeng; Zhang, Lifeng; Li, Wanfeng; Han, Suying; Yang, Wenhua; Qi, Liwang

    2013-01-01

    Quantitative real-time reverse transcription polymerase chain reaction (qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization. Caragana intermedia, a native desert shrub with strong drought-resistance, sand-fixing capacity and high forage value that is widespread in the desert land of west and northwest China, has not been investigated regarding the identification of reference genes suitable for the normalization of qPCR data. In this study, 10 candidate reference genes were analyzed in C. intermedia subjected to different abiotic (osmotic, salt, cold and heat) stresses, in two distinct plant organs (roots and leaves). The expression stability of these genes was assessed using geNorm, NormFinder and BestKeeper algorithms. The best-ranked reference genes differed across the different sets of samples, but UNK2, PP2A and SAND were the most stable across all tested samples. UNK2 and SAND would be appropriate for normalizing gene expression data for salt-treated roots, whereas the combination of UNK2, SAND and EF-1α would be appropriate for salt-treated leaves. UNK1, UNK2 and PP2A would be appropriate for PEG-treated (osmotic) roots, whereas the combination of TIP41 and PP2A was the most suitable for PEG-treated leaves. SAND, PP2A and TIP41 exhibited the most stable expression in heat-treated leaves. In cold-treated leaves, SAND and EF-1α were the most stably expressed. To further validate the suitability of the reference genes identified in this study, the expression levels of DREB1 and DREB2 (homologs of AtDREB1 and AtDREB2) were studied in parallel. This study is the first systematic analysis for the selection of superior reference genes for qPCR in C. intermedia under different abiotic stress conditions, and will benefit future studies on gene expression in C. intermedia and other species of the leguminous genus Caragana.

  10. Validation of reference genes from Eucalyptus spp. under different stress conditions

    PubMed Central

    2012-01-01

    Background The genus Eucalyptus consists of approximately 600 species and subspecies and has a physiological plasticity that allows some species to propagate in different regions of the world. Eucalyptus is a major source of cellulose for paper manufacturing, and its cultivation is limited by weather conditions, particularly water stress and low temperatures. Gene expression studies using quantitative reverse transcription polymerase chain reaction (qPCR) require reference genes, which must have stable expression to facilitate the comparison of the results from analyses using different species, tissues, and treatments. Such studies have been limited in eucalyptus. Results Eucalyptus globulus Labill, Eucalyptus urograndis (hybrid from Eucalyptus urophylla S.T. Blake X Eucalyptus grandis Hill ex-Maiden) and E. uroglobulus (hybrid from E. urograndis X E. globulus) were subjected to different treatments, including water deficiency and stress recovery, low temperatures, presence or absence of light, and their respective controls. Except for treatment with light, which examined the seedling hypocotyl or apical portion of the stem, the expression analyses were conducted in the apical and basal parts of the stem. To select the best pair of genes, the bioinformatics tools GeNorm and NormFinder were compared. Comprehensive analyses that did not differentiate between species, treatments, or tissue types, showed that IDH (isocitrate dehydrogenase), SAND (SAND protein), ACT (actin), and A-Tub (α-tubulin) genes were the most stable. IDH was the most stable gene in all of the treatments. Conclusion Comparing these results with those of other studies on eucalyptus, we concluded that five genes are stable in different species and experimental conditions: IDH, SAND, ACT, A-Tub, and UBQ (ubiquitin). It is usually recommended a minimum of two reference genes is expression analysis; therefore, we propose that IDH and two others genes among the five identified genes in this study

  11. The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.

    PubMed

    Cai, Jing; Li, Tao; Huang, Bangxing; Cheng, Henghui; Ding, Hui; Dong, Weihong; Xiao, Man; Liu, Ling; Wang, Zehua

    2014-01-01

    Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

  12. Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data

    PubMed Central

    2010-01-01

    Background Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes. Results By the use of two distinct algorithms, implemented by geNorm and NormFinder, we have assessed the gene expression of nine candidate reference genes in cotton: GhACT4, GhEF1α5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, GhβTUB3 and GhUBQ14. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of GhPP2A1 and GhUBQ14 genes were the most stable across all samples and also when distinct plants organs are examined. GhACT4 and GhUBQ14 present more stable expression during flower development, GhACT4 and GhFBX6 in the floral verticils and GhMZA and GhPTB during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development. Conclusion We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of GhUBQ14 and GhPP2A1 housekeeping genes as superior references for normalization of gene

  13. Validation of Reference Genes for Relative Quantitative Gene Expression Studies in Cassava (Manihot esculenta Crantz) by Using Quantitative Real-Time PCR.

    PubMed

    Hu, Meizhen; Hu, Wenbin; Xia, Zhiqiang; Zhou, Xincheng; Wang, Wenquan

    2016-01-01

    Reverse transcription quantitative real-time polymerase chain reaction (real-time PCR, also referred to as quantitative RT-PCR or RT-qPCR) is a highly sensitive and high-throughput method used to study gene expression. Despite the numerous advantages of RT-qPCR, its accuracy is strongly influenced by the stability of internal reference genes used for normalizations. To date, few studies on the identification of reference genes have been performed on cassava (Manihot esculenta Crantz). Therefore, we selected 26 candidate reference genes mainly via the three following channels: reference genes used in previous studies on cassava, the orthologs of the most stable Arabidopsis genes, and the sequences obtained from 32 cassava transcriptome sequence data. Then, we employed ABI 7900 HT and SYBR Green PCR mix to assess the expression of these genes in 21 materials obtained from various cassava samples under different developmental and environmental conditions. The stability of gene expression was analyzed using two statistical algorithms, namely geNorm and NormFinder. geNorm software suggests the combination of cassava4.1_017977 and cassava4.1_006391 as sufficient reference genes for major cassava samples, the union of cassava4.1_014335 and cassava4.1_006884 as best choice for drought stressed samples, and the association of cassava4.1_012496 and cassava4.1_006391 as optimal choice for normally grown samples. NormFinder software recommends cassava4.1_006884 or cassava4.1_006776 as superior reference for qPCR analysis of different materials and organs of drought stressed or normally grown cassava, respectively. Results provide an important resource for cassava reference genes under specific conditions. The limitations of these findings were also discussed. Furthermore, we suggested some strategies that may be used to select candidate reference genes.

  14. Validation of Reference Genes for Relative Quantitative Gene Expression Studies in Cassava (Manihot esculenta Crantz) by Using Quantitative Real-Time PCR

    PubMed Central

    Hu, Meizhen; Hu, Wenbin; Xia, Zhiqiang; Zhou, Xincheng; Wang, Wenquan

    2016-01-01

    Reverse transcription quantitative real-time polymerase chain reaction (real-time PCR, also referred to as quantitative RT-PCR or RT-qPCR) is a highly sensitive and high-throughput method used to study gene expression. Despite the numerous advantages of RT-qPCR, its accuracy is strongly influenced by the stability of internal reference genes used for normalizations. To date, few studies on the identification of reference genes have been performed on cassava (Manihot esculenta Crantz). Therefore, we selected 26 candidate reference genes mainly via the three following channels: reference genes used in previous studies on cassava, the orthologs of the most stable Arabidopsis genes, and the sequences obtained from 32 cassava transcriptome sequence data. Then, we employed ABI 7900 HT and SYBR Green PCR mix to assess the expression of these genes in 21 materials obtained from various cassava samples under different developmental and environmental conditions. The stability of gene expression was analyzed using two statistical algorithms, namely geNorm and NormFinder. geNorm software suggests the combination of cassava4.1_017977 and cassava4.1_006391 as sufficient reference genes for major cassava samples, the union of cassava4.1_014335 and cassava4.1_006884 as best choice for drought stressed samples, and the association of cassava4.1_012496 and cassava4.1_006391 as optimal choice for normally grown samples. NormFinder software recommends cassava4.1_006884 or cassava4.1_006776 as superior reference for qPCR analysis of different materials and organs of drought stressed or normally grown cassava, respectively. Results provide an important resource for cassava reference genes under specific conditions. The limitations of these findings were also discussed. Furthermore, we suggested some strategies that may be used to select candidate reference genes. PMID:27242878

  15. Reference Gene Selection for RT-qPCR Analysis of Flower Development in Chrysanthemum morifolium and Chrysanthemum lavandulifolium

    PubMed Central

    Qi, Shuai; Yang, Liwen; Wen, Xiaohui; Hong, Yan; Song, Xuebin; Zhang, Mengmeng; Dai, Silan

    2016-01-01

    Quantitative real-time PCR (qPCR) is a popular and powerful tool used to understand the molecular mechanisms of flower development. However, the accuracy of this approach depends on the stability of reference genes. The capitulum of chrysanthemums is very special, which is consisting of ray florets and disc florets. There are obvious differences between the two types of florets in symmetry, gender, histological structure, and function. Furthermore, the ray florets have various shapes. The objective of present study was to identify the stable reference genes in Chrysanthemum morifolium and Chrysanthemum lavandulifolium during the process of flower development. In this study, nine candidate reference genes were selected and evaluated for their expression stability acrosssamples during the process of flower development, and their stability was validated by four different algorithms (Bestkeeper, NormFinder, GeNorm, and Ref-finder). SAND (SAND family protein) was found to be the most stably expressed gene in all samples or different tissues during the process of C. lavandulifolium development. Both SAND and PGK (phosphoglycerate kinase) performed most stable in Chinese large-flowered chrysanthemum cultivars, and PGK was the best in potted chrysanthemums. There were differences in best reference genes among varieties as the genetic background of them were complex. These studies provide guidance for selecting reference genes for analyzing the expression pattern of floral development genes in chrysanthemums. PMID:27014310

  16. Identification of cell-specific patterns of reference gene stability in quantitative reverse-transcriptase polymerase chain reaction studies of embryonic, placental and neural stem models of prenatal ethanol exposure.

    PubMed

    Carnahan, Mindy N; Veazey, Kylee J; Muller, Daria; Tingling, Joseph D; Miranda, Rajesh C; Golding, Michael C

    2013-03-01

    Identification of the transcriptional networks disrupted by prenatal ethanol exposure remains a core requirement to better understanding the molecular mechanisms of alcohol-induced teratogenesis. In this regard, quantitative reverse-transcriptase polymerase chain reaction (qPCR) has emerged as an essential technique in our efforts to characterize alterations in gene expression brought on by exposure to alcohol. However, many publications continue to report the utilization of inappropriate methods of qPCR normalization, and for many in vitro models, no consistent set of empirically tested normalization controls have been identified. In the present study, we sought to identify a group of candidate reference genes for use within studies of alcohol exposed embryonic, placental, and neurosphere stem cells under both conditions maintaining stemness as well as throughout in vitro differentiation. To this end, we surveyed the recent literature and compiled a short list of fourteen candidate genes commonly used as normalization controls in qPCR studies of gene expression. This list included: Actb, B2m, Gapdh, Gusb, H2afz, Hk2, Hmbs, Hprt, Mrpl1, Pgk1, Ppia, Sdha, Tbp, and Ywhaz. From these studies, we find no single candidate gene was consistently refractory to the influence of alcohol nor completely stable throughout in vitro differentiation. Accordingly, we propose normalizing qPCR measurements to the geometric mean C(T) values obtained for three independent reference mRNAs as a reliable method to accurately interpret qPCR data and assess alterations in gene expression within alcohol treated cultures. Highlighting the importance of careful and empirical reference gene selection, the commonly used reference gene Actb was often amongst the least stable candidate genes tested. In fact, it would not serve as a valid normalization control in many cases. Data presented here will aid in the design of future experiments using stem cells to study the transcriptional processes

  17. Reference genes for accessing differential expression among developmental stages and analysis of differential expression of OBP genes in Anastrepha obliqua

    PubMed Central

    Nakamura, Aline Minali; Chahad-Ehlers, Samira; Lima, André Luís A.; Taniguti, Cristiane Hayumi; Sobrinho Jr., Iderval; Torres, Felipe Rafael; de Brito, Reinaldo Alves

    2016-01-01

    The West Indian fruit fly, Anastrepha obliqua, is an important agricultural pest in the New World. The use of pesticide-free methods to control invasive species such as this reinforces the search for genes potentially useful in their genetic control. Therefore, the study of chemosensory proteins involved with a range of responses to the chemical environment will help not only on the understanding of the species biology but may also help the development of environmentally friendly pest control strategies. Here we analyzed the expression patterns of three OBP genes, Obp19d_2, Obp56a and Obp99c, across different phases of A. obliqua development by qPCR. In order to do so, we tested eight and identified three reference genes for data normalization, rpl17, rpl18 and ef1a, which displayed stability for the conditions here tested. All OBPs showed differential expression on adults and some differential expression among adult stages. Obp99c had an almost exclusive expression in males and Obp56a showed high expression in virgin females. Thereby, our results provide relevant data not only for other gene expression studies in this species, as well as for the search of candidate genes that may help in the development of new pest control strategies. PMID:26818909

  18. Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata

    PubMed Central

    Zhao, Hongxi; Liu, Junlong; Li, Youquan; Yang, Congshan; Zhao, Shuaiyang; Liu, Juan; Liu, Aihong; Liu, Guangyuan; Yin, Hong; Guan, Guiquan; Luo, Jianxun

    2016-01-01

    Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (β-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata. PMID:26951977

  19. Efficient Computation of Approximate Gene Clusters Based on Reference Occurrences

    NASA Astrophysics Data System (ADS)

    Jahn, Katharina

    Whole genome comparison based on the analysis of gene cluster conservation has become a popular approach in comparative genomics. While gene order and gene content as a whole randomize over time, it is observed that certain groups of genes which are often functionally related remain co-located across species. However, the conservation is usually not perfect which turns the identification of these structures, often referred to as approximate gene clusters, into a challenging task. In this paper, we present a polynomial time algorithm that computes approximate gene clusters based on reference occurrences. We show that our approach yields highly comparable results to a more general approach and allows for approximate gene cluster detection in parameter ranges currently not feasible for non-reference based approaches.

  20. Combining qPCR and functional gene microarrays to directly link changes in the expression of the nirS gene to denitrification rates in aquatic sediment mesocosms

    NASA Astrophysics Data System (ADS)

    Bowen, J. L.; Babbin, A. R.; Ward, B. B.

    2010-12-01

    Molecular methods for the investigation of biogeochemical processes, including denitrification, are being developed at an astonishing rate, but it remains difficult to use the molecular information to understand the regulation and variation in biogeochemical transformation rates. By combining information on gene abundance and expression for nirS, a key gene in denitrification, with quantitative modeling of nitrogen fluxes, we can begin to understand the scales on which genetic signals vary in space and time, and how they relate to biogeochemical function. We used quantitative PCR, a functional gene microarray, and biogeochemical modeling to assess how denitrifier community composition (evaluated by DNA and cDNA of the nirS gene) changed over time in estuarine sediment mesocosms. Sediments and water were collected from coastal Massachusetts and maintained in replicated 20 L mesocosm experiments for 45 days. Sediments were collected for microbial analysis at weekly intervals throughout the experiment. Concentrations of all major nitrogen species were measured daily and used to derive rates of nitrification and denitrification from a Monte Carlo-based nonnegative least-squares analysis of finite difference equations. Denitrification rates peaked between day 18 and day 22, slightly after the peaks in nitrite concentration that were generated from oxidization of remineralized ammonium. In most mesocosms the peak in denitrification rates coincided with the peak in nirS gene abundance (DNA). Peaks in the expression of the nirS gene (cDNA), however, did not always correlate with peaks in the denitrification rates. The nirS microarray contained 39 archetype probes, three of which accounted for more than 60% of the DNA hybridization signal. Two of these clades also dominated the hybridization signal in cDNA, indicating that those organisms that are actively expressing nirS are not always the dominant members of the community. Fifteen of the 39 probes accounted for less than

  1. Reference genes for normalizing transcription in diploid and tetraploid Arabidopsis.

    PubMed

    Wang, Haibin; Wang, Jingjing; Jiang, Jiafu; Chen, Sumei; Guan, Zhiyong; Liao, Yuan; Chen, Fadi

    2014-10-27

    Published transcription data from a set of 19 diploid Arabidopsis thaliana and 5 tetraploid (3 allo- and 2 auto- tetraploid) Arabidopsis accessions were re-analysed to identify reliable reference genes for normalization purposes. Five conventional and 16 novel reference genes previously derived from microarray data covering a wide range of abundance in absolute expression levels in diploid A. thaliana Col-0 were employed. Transcript abundance was well conserved for all 21 potential reference genes in the diploid A. thaliana accessions, with geNorm and NormFinder analysis indicating that AT5G46630, AT1G13320, AT4G26410, AT5G60390 and AT5G08290 were the most stable. However, conservation was less good among the tetraploid accessions, with the transcription of seven of the 21 genes being undetectable in all allotetraploids. The most stable gene was AT5G46630, while AT1G13440 was the unstable one. Hence, the choice of reference gene(s) for A. thaliana is quite wide, but with respect to the analysis of transcriptomic data derived from the tetraploids, it is probably necessary to select more than one reference gene.

  2. Determining the optimal number of individual samples to pool for quantification of average herd levels of antimicrobial resistance genes in Danish pig herds using high-throughput qPCR.

    PubMed

    Clasen, Julie; Mellerup, Anders; Olsen, John Elmerdahl; Angen, Øystein; Folkesson, Anders; Halasa, Tariq; Toft, Nils; Birkegård, Anna Camilla

    2016-06-30

    The primary objective of this study was to determine the minimum number of individual fecal samples to pool together in order to obtain a representative sample for herd level quantification of antimicrobial resistance (AMR) genes in a Danish pig herd, using a novel high-throughput qPCR assay. The secondary objective was to assess the agreement between different methods of sample pooling. Quantification of AMR was achieved using a high-throughput qPCR method to quantify the levels of seven AMR genes (ermB, ermF, sulI, sulII, tet(M), tet(O) and tet(W)). A large variation in the levels of AMR genes was found between individual samples. As the number of samples in a pool increased, a decrease in sample variation was observed. It was concluded that the optimal pooling size is five samples, as an almost steady state in the variation was observed when pooling this number of samples. Good agreement between different pooling methods was found and the least time-consuming method of pooling, by transferring feces from each individual sample to a tube using a 10μl inoculation loop and adding 3.5ml of PBS, approximating a 10% solution, can therefore be used in future studies.

  3. Evaluation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in Pyrus pyrifolia using different tissue samples and seasonal conditions.

    PubMed

    Imai, Tsuyoshi; Ubi, Benjamin E; Saito, Takanori; Moriguchi, Takaya

    2014-01-01

    We have evaluated suitable reference genes for real time (RT)-quantitative PCR (qPCR) analysis in Japanese pear (Pyrus pyrifolia). We tested most frequently used genes in the literature such as β-Tubulin, Histone H3, Actin, Elongation factor-1α, Glyceraldehyde-3-phosphate dehydrogenase, together with newly added genes Annexin, SAND and TIP41. A total of 17 primer combinations for these eight genes were evaluated using cDNAs synthesized from 16 tissue samples from four groups, namely: flower bud, flower organ, fruit flesh and fruit skin. Gene expression stabilities were analyzed using geNorm and NormFinder software packages or by ΔCt method. geNorm analysis indicated three best performing genes as being sufficient for reliable normalization of RT-qPCR data. Suitable reference genes were different among sample groups, suggesting the importance of validation of gene expression stability of reference genes in the samples of interest. Ranking of stability was basically similar between geNorm and NormFinder, suggesting usefulness of these programs based on different algorithms. ΔCt method suggested somewhat different results in some groups such as flower organ or fruit skin; though the overall results were in good correlation with geNorm or NormFinder. Gene expression of two cold-inducible genes PpCBF2 and PpCBF4 were quantified using the three most and the three least stable reference genes suggested by geNorm. Although normalized quantities were different between them, the relative quantities within a group of samples were similar even when the least stable reference genes were used. Our data suggested that using the geometric mean value of three reference genes for normalization is quite a reliable approach to evaluating gene expression by RT-qPCR. We propose that the initial evaluation of gene expression stability by ΔCt method, and subsequent evaluation by geNorm or NormFinder for limited number of superior gene candidates will be a practical way of finding out

  4. A selection of reference genes and early-warning mRNA biomarkers for environmental monitoring using Mytilus spp. as sentinel species.

    PubMed

    Lacroix, C; Coquillé, V; Guyomarch, J; Auffret, M; Moraga, D

    2014-09-15

    mRNA biomarkers are promising tools for environmental health assessment and reference genes are needed to perform relevant qPCR analyses in tissue samples of sentinel species. In the present study, potential reference genes and mRNA biomarkers were tested in the gills and digestive glands of native and caged mussels (Mytilus spp.) exposed to harbor pollution. Results highlighted the difficulty to find stable reference genes in wild, non-model species and suggested the use of normalization indices instead of single genes as they exhibit a higher stability. Several target genes were found differentially expressed between mussel groups, especially in gills where cyp32, π-gst and CuZn-sod mRNA levels could be biomarker candidates. Multivariate analyses confirmed the ability of mRNA levels to highlight site-effects and suggested the use of several combined markers instead of individual ones. These findings support the use of qPCR technology and mRNA levels as early-warning biomarkers in marine monitoring programs.

  5. Synthetic certified DNA reference material for analysis of human erythropoietin transgene and transcript in gene doping and gene therapy.

    PubMed

    Baoutina, A; Bhat, S; Zheng, M; Partis, L; Dobeson, M; Alexander, I E; Emslie, K R

    2016-10-01

    There is a recognised need for standardisation of protocols for vector genome analysis used in vector manufacturing, to establish dosage, in biodistribution studies and to detect gene doping in sport. Analysis of vector genomes and transgene expression is typically performed by qPCR using plasmid-based calibrants incorporating transgenic sequences. These often undergo limited characterisation and differ between manufacturers, potentially leading to inaccurate quantification, inconsistent inter-laboratory results and affecting clinical outcomes. Contamination of negative samples with such calibrants could cause false positive results. We developed a design strategy for synthetic reference materials (RMs) with modified transgenic sequences to prevent false positives due to cross-contamination. When such RM is amplified in transgene-specific assays, the amplicons are distinguishable from transgene's amplicons based on size and sequence. Using human erythropoietin as a model, we produced certified RM according to this strategy and following ISO Guide 35. Using non-viral and viral vectors, we validated the effectiveness of this RM in vector genome analysis in blood in vitro. The developed design strategy could be applied to production of RMs for other transgenes, genes or transcripts. Together with validated PCR assays, such RMs form a measurement tool that facilitates standardised, accurate and reliable genetic analysis in various applications.

  6. Validation of reference genes for gene expression studies in soybean aphid, Aphis glycines Matsumura

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative real-time PCR (qRT-PCR) is a common tool for quantifying mRNA transcripts. To normalize results, a reference gene is mandatory. Aphis glycines is a significant soybean pest, yet gene expression and functional genomics studies are hindered by a lack of stable reference genes. We evalu...

  7. Laser capture microdissection of intestinal tissue from sea bass larvae using an optimized RNA integrity assay and validated reference genes

    PubMed Central

    Schaeck, M.; De Spiegelaere, W.; De Craene, J.; Van den Broeck, W.; De Spiegeleer, B.; Burvenich, C.; Haesebrouck, F.; Decostere, A.

    2016-01-01

    The increasing demand for a sustainable larviculture has promoted research regarding environmental parameters, diseases and nutrition, intersecting at the mucosal surface of the gastrointestinal tract of fish larvae. The combination of laser capture microdissection (LCM) and gene expression experiments allows cell specific expression profiling. This study aimed at optimizing an LCM protocol for intestinal tissue of sea bass larvae. Furthermore, a 3′/5′ integrity assay was developed for LCM samples of fish tissue, comprising low RNA concentrations. Furthermore, reliable reference genes for performing qPCR in larval sea bass gene expression studies were identified, as data normalization is critical in gene expression experiments using RT-qPCR. We demonstrate that a careful optimization of the LCM procedure allows recovery of high quality mRNA from defined cell populations in complex intestinal tissues. According to the geNorm and Normfinder algorithms, ef1a, rpl13a, rps18 and faua were the most stable genes to be implemented as reference genes for an appropriate normalization of intestinal tissue from sea bass across a range of experimental settings. The methodology developed here, offers a rapid and valuable approach to characterize cells/tissues in the intestinal tissue of fish larvae and their changes following pathogen exposure, nutritional/environmental changes, probiotic supplementation or a combination thereof. PMID:26883391

  8. Laser capture microdissection of intestinal tissue from sea bass larvae using an optimized RNA integrity assay and validated reference genes.

    PubMed

    Schaeck, M; De Spiegelaere, W; De Craene, J; Van den Broeck, W; De Spiegeleer, B; Burvenich, C; Haesebrouck, F; Decostere, A

    2016-02-17

    The increasing demand for a sustainable larviculture has promoted research regarding environmental parameters, diseases and nutrition, intersecting at the mucosal surface of the gastrointestinal tract of fish larvae. The combination of laser capture microdissection (LCM) and gene expression experiments allows cell specific expression profiling. This study aimed at optimizing an LCM protocol for intestinal tissue of sea bass larvae. Furthermore, a 3'/5' integrity assay was developed for LCM samples of fish tissue, comprising low RNA concentrations. Furthermore, reliable reference genes for performing qPCR in larval sea bass gene expression studies were identified, as data normalization is critical in gene expression experiments using RT-qPCR. We demonstrate that a careful optimization of the LCM procedure allows recovery of high quality mRNA from defined cell populations in complex intestinal tissues. According to the geNorm and Normfinder algorithms, ef1a, rpl13a, rps18 and faua were the most stable genes to be implemented as reference genes for an appropriate normalization of intestinal tissue from sea bass across a range of experimental settings. The methodology developed here, offers a rapid and valuable approach to characterize cells/tissues in the intestinal tissue of fish larvae and their changes following pathogen exposure, nutritional/environmental changes, probiotic supplementation or a combination thereof.

  9. Evaluation of putative reference genes for quantitative real-time PCR normalization in Lilium regale during development and under stress

    PubMed Central

    Zhang, Ming-Fang

    2016-01-01

    Normalization to reference genes is the most common method to avoid bias in real-time quantitative PCR (qPCR), which has been widely used for quantification of gene expression. Despite several studies on gene expression, Lilium, and particularly L. regale, has not been fully investigated regarding the evaluation of reference genes suitable for normalization. In this study, nine putative reference genes, namely 18S rRNA, ACT, BHLH, CLA, CYP, EF1, GAPDH, SAND and TIP41, were analyzed for accurate quantitative PCR normalization at different developmental stages and under different stress conditions, including biotic (Botrytis elliptica), drought, salinity, cold and heat stress. All these genes showed a wide variation in their Cq (quantification Cycle) values, and their stabilities were calculated by geNorm, NormFinder and BestKeeper. In a combination of the results from the three algorithms, BHLH was superior to the other candidates when all the experimental treatments were analyzed together; CLA and EF1 were also recommended by two of the three algorithms. As for specific conditions, EF1 under various developmental stages, SAND under biotic stress, CYP/GAPDH under drought stress, and TIP41 under salinity stress were generally considered suitable. All the algorithms agreed on the stability of SAND and GAPDH under cold stress, while only CYP was selected under heat stress by all of them. Additionally, the selection of optimal reference genes under biotic stress was further verified by analyzing the expression level of LrLOX in leaves inoculated with B. elliptica. Our study would be beneficial for future studies on gene expression and molecular breeding of Lilium. PMID:27019788

  10. Identification and validation of reference genes for accurate normalization of real-time quantitative PCR data in kiwifruit.

    PubMed

    Ferradás, Yolanda; Rey, Laura; Martínez, Óscar; Rey, Manuel; González, Ma Victoria

    2016-05-01

    Identification and validation of reference genes are required for the normalization of qPCR data. We studied the expression stability produced by eight primer pairs amplifying four common genes used as references for normalization. Samples representing different tissues, organs and developmental stages in kiwifruit (Actinidia chinensis var. deliciosa (A. Chev.) A. Chev.) were used. A total of 117 kiwifruit samples were divided into five sample sets (mature leaves, axillary buds, stigmatic arms, fruit flesh and seeds). All samples were also analysed as a single set. The expression stability of the candidate primer pairs was tested using three algorithms (geNorm, NormFinder and BestKeeper). The minimum number of reference genes necessary for normalization was also determined. A unique primer pair was selected for amplifying the 18S rRNA gene. The primer pair selected for amplifying the ACTIN gene was different depending on the sample set. 18S 2 and ACT 2 were the candidate primer pairs selected for normalization in the three sample sets (mature leaves, fruit flesh and stigmatic arms). 18S 2 and ACT 3 were the primer pairs selected for normalization in axillary buds. No primer pair could be selected for use as the reference for the seed sample set. The analysis of all samples in a single set did not produce the selection of any stably expressing primer pair. Considering data previously reported in the literature, we validated the selected primer pairs amplifying the FLOWERING LOCUS T gene for use in the normalization of gene expression in kiwifruit.

  11. Evaluating precision and accuracy when quantifying different endogenous control reference genes in maize using real-time PCR.

    PubMed

    Scholdberg, Tandace A; Norden, Tim D; Nelson, Daishia D; Jenkins, G Ronald

    2009-04-08

    The agricultural biotechnology industry routinely utilizes real-time quantitative PCR (RT-qPCR) for the detection of biotechnology-derived traits in plant material, particularly for meeting the requirements of legislative mandates that rely upon the trace detection of DNA. Quantification via real-time RT-qPCR in plant species involves the measurement of the copy number of a taxon-specific, endogenous control gene exposed to the same manipulations as the target gene prior to amplification. The International Organization for Standardization (ISO 21570:2005) specifies that the copy number of an endogenous reference gene be used for normalizing the concentration (expressed as a % w/w) of a trait-specific target gene when using RT-qPCR. For this purpose, the copy number of a constitutively expressed endogenous reference gene in the same sample is routinely monitored. Real-time qPCR was employed to evaluate the predictability and performance of commonly used endogenous control genes (starch synthase, SSIIb-2, SSIIb-3; alcohol dehydrogenase, ADH; high-mobility group, HMG; zein; and invertase, IVR) used to detect biotechnology-derived traits in maize. The data revealed relatively accurate and precise amplification efficiencies when isogenic maize was compared to certified reference standards, but highly variable results when 23 nonisogenic maize cultivars were compared to an IRMM Bt-11 reference standard. Identifying the most suitable endogenous control gene, one that amplifies consistently and predictably across different maize cultivars, and implementing this as an internationally recognized standard would contribute toward harmonized testing of biotechnology-derived traits in maize.

  12. Selection of reference genes for quantitative real-time PCR normalization in Panax ginseng at different stages of growth and in different organs.

    PubMed

    Liu, Jing; Wang, Qun; Sun, Minying; Zhu, Linlin; Yang, Michael; Zhao, Yu

    2014-01-01

    Quantitative real-time reverse transcription PCR (qRT-PCR) has become a widely used method for gene expression analysis; however, its data interpretation largely depends on the stability of reference genes. The transcriptomics of Panax ginseng, one of the most popular and traditional ingredients used in Chinese medicines, is increasingly being studied. Furthermore, it is vital to establish a series of reliable reference genes when qRT-PCR is used to assess the gene expression profile of ginseng. In this study, we screened out candidate reference genes for ginseng using gene expression data generated by a high-throughput sequencing platform. Based on the statistical tests, 20 reference genes (10 traditional housekeeping genes and 10 novel genes) were selected. These genes were tested for the normalization of expression levels in five growth stages and three distinct plant organs of ginseng by qPCR. These genes were subsequently ranked and compared according to the stability of their expressions using geNorm, NormFinder, and BestKeeper computational programs. Although the best reference genes were found to vary across different samples, CYP and EF-1α were the most stable genes amongst all samples. GAPDH/30S RPS20, CYP/60S RPL13 and CYP/QCR were the optimum pair of reference genes in the roots, stems, and leaves. CYP/60S RPL13, CYP/eIF-5A, aTUB/V-ATP, eIF-5A/SAR1, and aTUB/pol IIa were the most stably expressed combinations in each of the five developmental stages. Our study serves as a foundation for developing an accurate method of qRT-PCR and will benefit future studies on gene expression profiles of Panax Ginseng.

  13. Evaluation of Sorghum [Sorghum bicolor (L.)] Reference Genes in Various Tissues and under Abiotic Stress Conditions for Quantitative Real-Time PCR Data Normalization.

    PubMed

    Sudhakar Reddy, Palakolanu; Srinivas Reddy, Dumbala; Sivasakthi, Kaliamoorthy; Bhatnagar-Mathur, Pooja; Vadez, Vincent; Sharma, Kiran K

    2016-01-01

    Accurate and reliable gene expression data from qPCR depends on stable reference gene expression for potential gene functional analyses. In this study, 15 reference genes were selected and analyzed in various sample sets including abiotic stress treatments (salt, cold, water stress, heat, and abscisic acid) and tissues (leaves, roots, seedlings, panicle, and mature seeds). Statistical tools, including geNorm, NormFinder and RefFinder, were utilized to assess the suitability of reference genes based on their stability rankings for various sample groups. For abiotic stress, PP2A and CYP were identified as the most stable genes. In contrast, EIF4α was the most stable in the tissue sample set, followed by PP2A; PP2A was the most stable in all the sample set, followed by EIF4α. GAPDH, and UBC1 were the least stably expressed in the tissue and all the sample sets. These results also indicated that the use of two candidate reference genes would be sufficient for the optimization of normalization studies. To further verify the suitability of these genes for use as reference genes, SbHSF5 and SbHSF13 gene expression levels were normalized using the most and least stable sorghum reference genes in root and water stressed-leaf tissues of five sorghum varieties. This is the first systematic study of the selection of the most stable reference genes for qPCR-related assays in Sorghum bicolor that will potentially benefit future gene expression studies in sorghum and other closely related species.

  14. Evaluation of Sorghum [Sorghum bicolor (L.)] Reference Genes in Various Tissues and under Abiotic Stress Conditions for Quantitative Real-Time PCR Data Normalization

    PubMed Central

    Sudhakar Reddy, Palakolanu; Srinivas Reddy, Dumbala; Sivasakthi, Kaliamoorthy; Bhatnagar-Mathur, Pooja; Vadez, Vincent; Sharma, Kiran K.

    2016-01-01

    Accurate and reliable gene expression data from qPCR depends on stable reference gene expression for potential gene functional analyses. In this study, 15 reference genes were selected and analyzed in various sample sets including abiotic stress treatments (salt, cold, water stress, heat, and abscisic acid) and tissues (leaves, roots, seedlings, panicle, and mature seeds). Statistical tools, including geNorm, NormFinder and RefFinder, were utilized to assess the suitability of reference genes based on their stability rankings for various sample groups. For abiotic stress, PP2A and CYP were identified as the most stable genes. In contrast, EIF4α was the most stable in the tissue sample set, followed by PP2A; PP2A was the most stable in all the sample set, followed by EIF4α. GAPDH, and UBC1 were the least stably expressed in the tissue and all the sample sets. These results also indicated that the use of two candidate reference genes would be sufficient for the optimization of normalization studies. To further verify the suitability of these genes for use as reference genes, SbHSF5 and SbHSF13 gene expression levels were normalized using the most and least stable sorghum reference genes in root and water stressed-leaf tissues of five sorghum varieties. This is the first systematic study of the selection of the most stable reference genes for qPCR-related assays in Sorghum bicolor that will potentially benefit future gene expression studies in sorghum and other closely related species. PMID:27200008

  15. Bacterial reference genes for gene expression studies by RT-qPCR: survey and analysis.

    PubMed

    Rocha, Danilo J P; Santos, Carolina S; Pacheco, Luis G C

    2015-09-01

    The appropriate choice of reference genes is essential for accurate normalization of gene expression data obtained by the method of reverse transcription quantitative real-time PCR (RT-qPCR). In 2009, a guideline called the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) highlighted the importance of the selection and validation of more than one suitable reference gene for obtaining reliable RT-qPCR results. Herein, we searched the recent literature in order to identify the bacterial reference genes that have been most commonly validated in gene expression studies by RT-qPCR (in the first 5 years following publication of the MIQE guidelines). Through a combination of different search parameters with the text mining tool MedlineRanker, we identified 145 unique bacterial genes that were recently tested as candidate reference genes. Of these, 45 genes were experimentally validated and, in most of the cases, their expression stabilities were verified using the software tools geNorm and NormFinder. It is noteworthy that only 10 of these reference genes had been validated in two or more of the studies evaluated. An enrichment analysis using Gene Ontology classifications demonstrated that genes belonging to the functional categories of DNA Replication (GO: 0006260) and Transcription (GO: 0006351) rendered a proportionally higher number of validated reference genes. Three genes in the former functional class were also among the top five most stable genes identified through an analysis of gene expression data obtained from the Pathosystems Resource Integration Center. These results may provide a guideline for the initial selection of candidate reference genes for RT-qPCR studies in several different bacterial species.

  16. Evaluation of Appropriate Reference Genes for Gene Expression Normalization during Watermelon Fruit Development.

    PubMed

    Kong, Qiusheng; Yuan, Jingxian; Gao, Lingyun; Zhao, Liqiang; Cheng, Fei; Huang, Yuan; Bie, Zhilong

    2015-01-01

    Gene expression analysis in watermelon (Citrullus lanatus) fruit has drawn considerable attention with the availability of genome sequences to understand the regulatory mechanism of fruit development and to improve its quality. Real-time quantitative reverse-transcription PCR (qRT-PCR) is a routine technique for gene expression analysis. However, appropriate reference genes for transcript normalization in watermelon fruits have not been well characterized. The aim of this study was to evaluate the appropriateness of 12 genes for their potential use as reference genes in watermelon fruits. Expression variations of these genes were measured in 48 samples obtained from 12 successive developmental stages of parthenocarpic and fertilized fruits of two watermelon genotypes by using qRT-PCR analysis. Considering the effects of genotype, fruit setting method, and developmental stage, geNorm determined clathrin adaptor complex subunit (ClCAC), β-actin (ClACT), and alpha tubulin 5 (ClTUA5) as the multiple reference genes in watermelon fruit. Furthermore, ClCAC alone or together with SAND family protein (ClSAND) was ranked as the single or two best reference genes by NormFinder. By using the top-ranked reference genes to normalize the transcript abundance of phytoene synthase (ClPSY1), a good correlation between lycopene accumulation and ClPSY1 expression pattern was observed in ripening watermelon fruit. These validated reference genes will facilitate the accurate measurement of gene expression in the studies on watermelon fruit biology.

  17. Validation of endogenous control reference genes for normalizing gene expression studies in endometrial carcinoma.

    PubMed

    Ayakannu, Thangesweran; Taylor, Anthony H; Willets, Jonathon M; Brown, Laurence; Lambert, David G; McDonald, John; Davies, Quentin; Moss, Esther L; Konje, Justin C

    2015-09-01

    Real-time quantitative RT-PCR (qRT-PCR) is a powerful technique used for the relative quantification of target genes, using reference (housekeeping) genes for normalization to ensure the generation of accurate and robust data. A systematic examination of the suitability of endogenous reference genes for gene expression studies in endometrial cancer tissues is absent. The aims of this study were therefore to identify and evaluate from the thirty-two possible reference genes from a TaqMan(®) array panel their suitability as an internal control gene. The mathematical software packages geNorm qBasePLUS identified Pumilio homolog 1 (Drosophila) (PUM1), ubiquitin C (UBC), phosphoglycerate kinase (PGK1), mitochondrial ribosomal protein L19 (MRPL19) and peptidylpropyl isomerase A (cyclophilin A) (PPIA) as the best reference gene combination, whilst NormFinder identified MRPL19 as the best single reference gene, with importin 8 (IPO8) and PPIA being the best combination of two reference genes. BestKeeper ranked MRPL19 as the most stably expressed gene. In addition, the study was validated by examining the relative expression of a test gene, which encodes the cannabinoid receptor 1 (CB1). A significant difference in CB1 mRNA expression between malignant and normal endometrium using MRPL19, PPIA, and IP08 in combination was observed. The use of MRPL19, IPO8 and PPIA was identified as the best reference gene combination for the normalization of gene expression levels in endometrial carcinoma. This study demonstrates that the arbitrary selection of endogenous control reference genes for normalization in qRT-PCR studies of endometrial carcinoma, without validation, risks the production of inaccurate data and should therefore be discouraged.

  18. Assessment of Suitable Reference Genes for Quantitative Gene Expression Studies in Melon Fruits

    PubMed Central

    Kong, Qiusheng; Gao, Lingyun; Cao, Lei; Liu, Yue; Saba, Hameed; Huang, Yuan; Bie, Zhilong

    2016-01-01

    Melon (Cucumis melo L.) is an attractive model plant for investigating fruit development because of its morphological, physiological, and biochemical diversity. Quantification of gene expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR) with stably expressed reference genes for normalization can effectively elucidate the biological functions of genes that regulate fruit development. However, the reference genes for data normalization in melon fruits have not yet been systematically validated. This study aims to assess the suitability of 20 genes for their potential use as reference genes in melon fruits. Expression variations of these genes were measured in 24 samples that represented different developmental stages of fertilized and parthenocarpic melon fruits by qRT-PCR analysis. GeNorm identified ribosomal protein L (CmRPL) and cytosolic ribosomal protein S15 (CmRPS15) as the best pair of reference genes, and as many as five genes including CmRPL, CmRPS15, TIP41-like family protein (CmTIP41), cyclophilin ROC7 (CmCYP7), and ADP ribosylation factor 1 (CmADP) were required for more reliable normalization. NormFinder ranked CmRPS15 as the best single reference gene, and RAN GTPase gene family (CmRAN) and TATA-box binding protein (CmTBP2) as the best combination of reference genes in melon fruits. Their effectiveness was further validated by parallel analyses on the activities of soluble acid invertase and sucrose phosphate synthase, and expression profiles of their respective encoding genes CmAIN2 and CmSPS1, as well as sucrose contents during melon fruit ripening. The validated reference genes will help to improve the accuracy of gene expression studies in melon fruits. PMID:27536316

  19. Reference-based gene model prediction on DNA contigs

    SciTech Connect

    Xu, Y.; Uberbacher, E.C.

    1997-01-01

    This paper presents an algorithm for constructing multiple gene models on a set of contigs of a large genomic clone. The algorithm first uses pattern recognition-based methods to locate exons or partial exons in each contig, and then applies protein homology or EST information from the databases, as reference models, to parse the predicted exons into gene models. In the phase of gene model construction, the algorithm uses a unified framework for genes ranging from situation with homologous proteins/ESTs to no homologous protein/EST in the database. By exploiting protein homology or EST information, the algorithm is able to (1) parse exons into multiple gene models over a set of DNA contigs (possibly unoriented and unordered); (2) remove falsely predicted exons; and (3) identify and locate exons missed by the initial exon prediction.

  20. Identification of suitable reference genes for gene expression studies of shoulder instability.

    PubMed

    Leal, Mariana Ferreira; Belangero, Paulo Santoro; Cohen, Carina; Figueiredo, Eduardo Antônio; Loyola, Leonor Casilla; Pochini, Alberto Castro; Smith, Marília Cardoso; Andreoli, Carlos Vicente; Belangero, Sintia Iole; Ejnisman, Benno; Cohen, Moises

    2014-01-01

    Shoulder instability is a common shoulder injury, and patients present with plastic deformation of the glenohumeral capsule. Gene expression analysis may be a useful tool for increasing the general understanding of capsule deformation, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an effective method for such studies. Although RT-qPCR is highly sensitive and specific, it requires the use of suitable reference genes for data normalization to guarantee meaningful and reproducible results. In the present study, we evaluated the suitability of a set of reference genes using samples from the glenohumeral capsules of individuals with and without shoulder instability. We analyzed the expression of six commonly used reference genes (ACTB, B2M, GAPDH, HPRT1, TBP and TFRC) in the antero-inferior, antero-superior and posterior portions of the glenohumeral capsules of cases and controls. The stability of the candidate reference gene expression was determined using four software packages: NormFinder, geNorm, BestKeeper and DataAssist. Overall, HPRT1 was the best single reference gene, and HPRT1 and B2M composed the best pair of reference genes from different analysis groups, including simultaneous analysis of all tissue samples. GenEx software was used to identify the optimal number of reference genes to be used for normalization and demonstrated that the accumulated standard deviation resulting from the use of 2 reference genes was similar to that resulting from the use of 3 or more reference genes. To identify the optimal combination of reference genes, we evaluated the expression of COL1A1. Although the use of different reference gene combinations yielded variable normalized quantities, the relative quantities within sample groups were similar and confirmed that no obvious differences were observed when using 2, 3 or 4 reference genes. Consequently, the use of 2 stable reference genes for normalization, especially HPRT1 and B2M, is a

  1. The Challenge of Stability in High-Throughput Gene Expression Analysis: Comprehensive Selection and Evaluation of Reference Genes for BALB/c Mice Spleen Samples in the Leishmania infantum Infection Model

    PubMed Central

    Gonzalez-García, Ana C.; Quispe-Ricalde, M. Antonieta; Larraga, Vicente; Valladares, Basilio; Carmelo, Emma

    2016-01-01

    The interaction of Leishmania with BALB/c mice induces dramatic changes in transcriptome patterns in the parasite, but also in the target organs (spleen, liver…) due to its response against infection. Real-time quantitative PCR (qPCR) is an interesting approach to analyze these changes and understand the immunological pathways that lead to protection or progression of disease. However, qPCR results need to be normalized against one or more reference genes (RG) to correct for non-specific experimental variation. The development of technical platforms for high-throughput qPCR analysis, and powerful software for analysis of qPCR data, have acknowledged the problem that some reference genes widely used due to their known or suspected “housekeeping” roles, should be avoided due to high expression variability across different tissues or experimental conditions. In this paper we evaluated the stability of 112 genes using three different algorithms: geNorm, NormFinder and RefFinder in spleen samples from BALB/c mice under different experimental conditions (control and Leishmania infantum-infected mice). Despite minor discrepancies in the stability ranking shown by the three methods, most genes show very similar performance as RG (either good or poor) across this massive data set. Our results show that some of the genes traditionally used as RG in this model (i.e. B2m, Polr2a and Tbp) are clearly outperformed by others. In particular, the combination of Il2rg + Itgb2 was identified among the best scoring candidate RG for every group of mice and every algorithm used in this experimental model. Finally, we have demonstrated that using “traditional” vs rationally-selected RG for normalization of gene expression data may lead to loss of statistical significance of gene expression changes when using large-scale platforms, and therefore misinterpretation of results. Taken together, our results highlight the need for a comprehensive, high-throughput search for the most

  2. The Challenge of Stability in High-Throughput Gene Expression Analysis: Comprehensive Selection and Evaluation of Reference Genes for BALB/c Mice Spleen Samples in the Leishmania infantum Infection Model.

    PubMed

    Hernandez-Santana, Yasmina E; Ontoria, Eduardo; Gonzalez-García, Ana C; Quispe-Ricalde, M Antonieta; Larraga, Vicente; Valladares, Basilio; Carmelo, Emma

    The interaction of Leishmania with BALB/c mice induces dramatic changes in transcriptome patterns in the parasite, but also in the target organs (spleen, liver…) due to its response against infection. Real-time quantitative PCR (qPCR) is an interesting approach to analyze these changes and understand the immunological pathways that lead to protection or progression of disease. However, qPCR results need to be normalized against one or more reference genes (RG) to correct for non-specific experimental variation. The development of technical platforms for high-throughput qPCR analysis, and powerful software for analysis of qPCR data, have acknowledged the problem that some reference genes widely used due to their known or suspected "housekeeping" roles, should be avoided due to high expression variability across different tissues or experimental conditions. In this paper we evaluated the stability of 112 genes using three different algorithms: geNorm, NormFinder and RefFinder in spleen samples from BALB/c mice under different experimental conditions (control and Leishmania infantum-infected mice). Despite minor discrepancies in the stability ranking shown by the three methods, most genes show very similar performance as RG (either good or poor) across this massive data set. Our results show that some of the genes traditionally used as RG in this model (i.e. B2m, Polr2a and Tbp) are clearly outperformed by others. In particular, the combination of Il2rg + Itgb2 was identified among the best scoring candidate RG for every group of mice and every algorithm used in this experimental model. Finally, we have demonstrated that using "traditional" vs rationally-selected RG for normalization of gene expression data may lead to loss of statistical significance of gene expression changes when using large-scale platforms, and therefore misinterpretation of results. Taken together, our results highlight the need for a comprehensive, high-throughput search for the most stable

  3. Real-Time Quantitative PCR (QPCR) and Reverse Transcription-QPCR for Detection and Enumeration of Total Yeasts in Wine▿

    PubMed Central

    Hierro, Núria; Esteve-Zarzoso, Braulio; González, Ángel; Mas, Albert; Guillamón, Jose M.

    2006-01-01

    Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage. PMID:17088381

  4. Real-time quantitative PCR (QPCR) and reverse transcription-QPCR for detection and enumeration of total yeasts in wine.

    PubMed

    Hierro, Núria; Esteve-Zarzoso, Braulio; González, Angel; Mas, Albert; Guillamón, Jose M

    2006-11-01

    Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage.

  5. The choice of reference genes for assessing gene expression in sugarcane under salinity and drought stresses.

    PubMed

    Guo, Jinlong; Ling, Hui; Wu, Qibin; Xu, Liping; Que, Youxiong

    2014-11-13

    Sugarcane (Saccharum spp. hybrids) is a world-wide cash crop for sugar and biofuel in tropical and subtropical regions and suffers serious losses in cane yield and sugar content under salinity and drought stresses. Although real-time quantitative PCR has a numerous advantage in the expression quantification of stress-related genes for the elaboration of the corresponding molecular mechanism in sugarcane, the variation happened across the process of gene expression quantification should be normalized and monitored by introducing one or several reference genes. To validate suitable reference genes or gene sets for sugarcane gene expression normalization, 13 candidate reference genes have been tested across 12 NaCl- and PEG-treated sugarcane samples for four sugarcane genotypes using four commonly used systematic statistical algorithms termed geNorm, BestKeeper, NormFinder and the deltaCt method. The results demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and eukaryotic elongation factor 1-alpha (eEF-1a) were identified as suitable reference genes for gene expression normalization under salinity/drought-treatment in sugarcane. Moreover, the expression analyses of SuSK and 6PGDH further validated that a combination of clathrin adaptor complex (CAC) and cullin (CUL) as reference should be better for gene expression normalization. These results can facilitate the future research on gene expression in sugarcane under salinity and drought stresses.

  6. Screening Reliable Reference Genes for RT-qPCR Analysis of Gene Expression in Moringa oleifera

    PubMed Central

    Deng, Li-Ting; Wu, Yu-Ling; Li, Jun-Cheng; OuYang, Kun-Xi; Ding, Mei-Mei; Zhang, Jun-Jie; Li, Shu-Qi; Lin, Meng-Fei; Chen, Han-Bin; Hu, Xin-Sheng; Chen, Xiao-Yang

    2016-01-01

    Moringa oleifera is a promising plant species for oil and forage, but its genetic improvement is limited. Our current breeding program in this species focuses on exploiting the functional genes associated with important agronomical traits. Here, we screened reliable reference genes for accurately quantifying the expression of target genes using the technique of real-time quantitative polymerase chain reaction (RT-qPCR) in M. oleifera. Eighteen candidate reference genes were selected from a transcriptome database, and their expression stabilities were examined in 90 samples collected from the pods in different developmental stages, various tissues, and the roots and leaves under different conditions (low or high temperature, sodium chloride (NaCl)- or polyethyleneglycol (PEG)- simulated water stress). Analyses with geNorm, NormFinder and BestKeeper algorithms revealed that the reliable reference genes differed across sample designs and that ribosomal protein L1 (RPL1) and acyl carrier protein 2 (ACP2) were the most suitable reference genes in all tested samples. The experiment results demonstrated the significance of using the properly validated reference genes and suggested the use of more than one reference gene to achieve reliable expression profiles. In addition, we applied three isotypes of the superoxide dismutase (SOD) gene that are associated with plant adaptation to abiotic stress to confirm the efficacy of the validated reference genes under NaCl and PEG water stresses. Our results provide a valuable reference for future studies on identifying important functional genes from their transcriptional expressions via RT-qPCR technique in M. oleifera. PMID:27541138

  7. Development of a qPCR Strategy to Select Bean Genes Involved in Plant Defense Response and Regulated by the Trichoderma velutinum - Rhizoctonia solani Interaction.

    PubMed

    Mayo, Sara; Cominelli, Eleonora; Sparvoli, Francesca; González-López, Oscar; Rodríguez-González, Alvaro; Gutiérrez, Santiago; Casquero, Pedro A

    2016-01-01

    Bean production is affected by a wide diversity of fungal pathogens, among them Rhizoctonia solani is one of the most important. A strategy to control bean infectious diseases, mainly those caused by fungi, is based on the use of biocontrol agents (BCAs) that can reduce the negative effects of plant pathogens and also can promote positive responses in the plant. Trichoderma is a fungal genus that is able to induce the expression of genes involved in plant defense response and also to promote plant growth, root development and nutrient uptake. In this article, a strategy that combines in silico analysis and real time PCR to detect additional bean defense-related genes, regulated by the presence of Trichoderma velutinum and/or R. solani has been applied. Based in this strategy, from the 48 bean genes initially analyzed, 14 were selected, and only WRKY33, CH5b and hGS showed an up-regulatory response in the presence of T. velutinum. The other genes were or not affected (OSM34) or down-regulated by the presence of this fungus. R. solani infection resulted in a down-regulation of most of the genes analyzed, except PR1, OSM34 and CNGC2 that were not affected, and the presence of both, T. velutinum and R. solani, up-regulates hGS and down-regulates all the other genes analyzed, except CH5b which was not significantly affected. As conclusion, the strategy described in the present work has been shown to be effective to detect genes involved in plant defense, which respond to the presence of a BCA or to a pathogen and also to the presence of both. The selected genes show significant homology with previously described plant defense genes and they are expressed in bean leaves of plants treated with T. velutinum and/or infected with R. solani.

  8. Development of a qPCR Strategy to Select Bean Genes Involved in Plant Defense Response and Regulated by the Trichoderma velutinum – Rhizoctonia solani Interaction

    PubMed Central

    Mayo, Sara; Cominelli, Eleonora; Sparvoli, Francesca; González-López, Oscar; Rodríguez-González, Alvaro; Gutiérrez, Santiago; Casquero, Pedro A.

    2016-01-01

    Bean production is affected by a wide diversity of fungal pathogens, among them Rhizoctonia solani is one of the most important. A strategy to control bean infectious diseases, mainly those caused by fungi, is based on the use of biocontrol agents (BCAs) that can reduce the negative effects of plant pathogens and also can promote positive responses in the plant. Trichoderma is a fungal genus that is able to induce the expression of genes involved in plant defense response and also to promote plant growth, root development and nutrient uptake. In this article, a strategy that combines in silico analysis and real time PCR to detect additional bean defense-related genes, regulated by the presence of Trichoderma velutinum and/or R. solani has been applied. Based in this strategy, from the 48 bean genes initially analyzed, 14 were selected, and only WRKY33, CH5b and hGS showed an up-regulatory response in the presence of T. velutinum. The other genes were or not affected (OSM34) or down-regulated by the presence of this fungus. R. solani infection resulted in a down-regulation of most of the genes analyzed, except PR1, OSM34 and CNGC2 that were not affected, and the presence of both, T. velutinum and R. solani, up-regulates hGS and down-regulates all the other genes analyzed, except CH5b which was not significantly affected. As conclusion, the strategy described in the present work has been shown to be effective to detect genes involved in plant defense, which respond to the presence of a BCA or to a pathogen and also to the presence of both. The selected genes show significant homology with previously described plant defense genes and they are expressed in bean leaves of plants treated with T. velutinum and/or infected with R. solani. PMID:27540382

  9. Identification of reference genes and validation for gene expression studies in diverse axolotl (Ambystoma mexicanum) tissues.

    PubMed

    Guelke, Eileen; Bucan, Vesna; Liebsch, Christina; Lazaridis, Andrea; Radtke, Christine; Vogt, Peter M; Reimers, Kerstin

    2015-04-10

    For the precise quantitative RT-PCR normalization a set of valid reference genes is obligatory. Moreover have to be taken into concern the experimental conditions as they bias the regulation of reference genes. Up till now, no reference targets have been described for the axolotl (Ambystoma mexicanum). In a search in the public database SalSite for genetic information of the axolotl we identified fourteen presumptive reference genes, eleven of which were further tested for their gene expression stability. This study characterizes the expressional patterns of 11 putative endogenous control genes during axolotl limb regeneration and in an axolotl tissue panel. All 11 reference genes showed variable expression. Strikingly, ACTB was to be found most stable expressed in all comparative tissue groups, so we reason it to be suitable for all different kinds of axolotl tissue-type investigations. Moreover do we suggest GAPDH and RPLP0 as suitable for certain axolotl tissue analysis. When it comes to axolotl limb regeneration, a validated pair of reference genes is ODC and RPLP0. With these findings, new insights into axolotl gene expression profiling might be gained.

  10. Reference genes for gene expression studies in wheat flag leaves grown under different farming conditions

    PubMed Central

    2011-01-01

    Background Internal control genes with highly uniform expression throughout the experimental conditions are required for accurate gene expression analysis as no universal reference genes exists. In this study, the expression stability of 24 candidate genes from Triticum aestivum cv. Cubus flag leaves grown under organic and conventional farming systems was evaluated in two locations in order to select suitable genes that can be used for normalization of real-time quantitative reverse-transcription PCR (RT-qPCR) reactions. The genes were selected among the most common used reference genes as well as genes encoding proteins involved in several metabolic pathways. Findings Individual genes displayed different expression rates across all samples assayed. Applying geNorm, a set of three potential reference genes were suitable for normalization of RT-qPCR reactions in winter wheat flag leaves cv. Cubus: TaFNRII (ferredoxin-NADP(H) oxidoreductase; AJ457980.1), ACT2 (actin 2; TC234027), and rrn26 (a putative homologue to RNA 26S gene; AL827977.1). In addition of these three genes that were also top-ranked by NormFinder, two extra genes: CYP18-2 (Cyclophilin A, AY456122.1) and TaWIN1 (14-3-3 like protein, AB042193) were most consistently stably expressed. Furthermore, we showed that TaFNRII, ACT2, and CYP18-2 are suitable for gene expression normalization in other two winter wheat varieties (Tommi and Centenaire) grown under three treatments (organic, conventional and no nitrogen) and a different environment than the one tested with cv. Cubus. Conclusions This study provides a new set of reference genes which should improve the accuracy of gene expression analyses when using wheat flag leaves as those related to the improvement of nitrogen use efficiency for cereal production. PMID:21951810

  11. Identification of reference genes in human myelomonocytic cells for gene expression studies in altered gravity.

    PubMed

    Thiel, Cora S; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Unverdorben, Felix; Buttron, Isabell; Lauber, Beatrice; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E; Ullrich, Oliver

    2015-01-01

    Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes ("housekeeping genes") are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

  12. Reference genes for gene expression analysis in proliferating and differentiating human keratinocytes.

    PubMed

    Lanzafame, Manuela; Botta, Elena; Teson, Massimo; Fortugno, Paola; Zambruno, Giovanna; Stefanini, Miria; Orioli, Donata

    2015-04-01

    Abnormalities in keratinocyte growth and differentiation have a pathogenic significance in many skin disorders and result in gene expression alterations detectable by quantitative real-time RT-PCR (qRT-PCR). Relative quantification based on endogenous control (EC) genes is the commonly adopted approach, and the use of multiple reference genes from independent pathways is considered a best practice guideline, unless fully validated EC genes are available. The literature on optimal reference genes during in vitro calcium-induced differentiation of normal human epidermal keratinocytes (NHEK) is inconsistent. In many studies, the expression of target genes is compared to that of housekeeping genes whose expression, however, significantly varies during keratinocyte differentiation. Here, we report the results of our investigations on the expression stability of 15 candidate EC genes, including those commonly used as reference in expression analysis by qRT-PCR, during NHEK calcium-induced differentiation. We demonstrate that YWHAZ and UBC are extremely stable genes, and therefore, they represent optimal EC genes for expression studies in proliferating and calcium-induced differentiating NHEK. Furthermore, we demonstrate that YWHAZ/14-3-3-zeta is a suitable reference for quantitative comparison of both transcript and protein levels.

  13. Identification of Reference Genes for Quantitative Real-Time PCR in Date Palm (Phoenix dactylifera L.) Subjected to Drought and Salinity

    PubMed Central

    V. Patankar, Himanshu; M. Assaha, Dekoum V.; Al-Yahyai, Rashid; Sunkar, Ramanjulu

    2016-01-01

    Date palm is an important crop plant in the arid and semi-arid regions supporting human population in the Middle East and North Africa. These areas have been largely affected by drought and salinity due to insufficient rainfall and improper irrigation practices. Date palm is a relatively salt- and drought-tolerant plant and more recently efforts have been directed to identifying genes and pathways that confer stress tolerance in this species. Quantitative real-time PCR (qPCR) is a promising technique for the analysis of stress-induced differential gene expression, which involves the use of stable reference genes for normalizing gene expression. In an attempt to find the best reference genes for date palm’s drought and salinity research, we evaluated the stability of 12 most commonly used reference genes using the geNorm, NormFinder, BestKeeper statistical algorithms and the comparative ΔCT method. The comprehensive results revealed that HEAT SHOCK PROTEIN (HSP), UBIQUITIN (UBQ) and YTH domain-containing family protein (YT521) were stable in drought-stressed leaves whereas GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH), ACTIN and TUBULIN were stable in drought-stressed roots. On the other hand, SMALL SUBUNIT RIBOSOMAL RNA (25S), YT521 and 18S ribosomal RNA (18S); and UBQ, ACTIN and ELONGATION FACTOR 1-ALPHA (eEF1a) were stable in leaves and roots, respectively, under salt stress. The stability of these reference genes was verified by using the abiotic stress-responsive CYTOSOLIC Cu/Zn SUPEROXIDE DISMUTASE (Cyt-Cu/Zn SOD), an ABA RECEPTOR, and a PROLINE TRANSPORTER 2 (PRO) genes. A combination of top 2 or 3 stable reference genes were found to be suitable for normalization of the target gene expression and will facilitate gene expression analysis studies aimed at identifying functional genes associated with drought and salinity tolerance in date palm. PMID:27824922

  14. Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don

    PubMed Central

    Xiao, Zheng; Sun, Xiaobo; Liu, Xiaoqing; Li, Chang; He, Lisi; Chen, Shangping; Su, Jiale

    2016-01-01

    The quantitative real-time polymerase chain reaction (qRT-PCR) approach has become a widely used method to analyze expression patterns of target genes. The selection of an optimal reference gene is a prerequisite for the accurate normalization of gene expression in qRT-PCR. The present study constitutes the first systematic evaluation of potential reference genes in Rhododendron molle G. Don. Eleven candidate reference genes in different tissues and flowers at different developmental stages of R. molle were assessed using the following three software packages: GeNorm, NormFinder, and BestKeeper. The results showed that EF1-α (elongation factor 1-alpha), 18S (18s ribosomal RNA), and RPL3 (ribosomal protein L3) were the most stable reference genes in developing rhododendron flowers and, thus, in all of the tested samples, while tublin (TUB) was the least stable. ACT5 (actin), RPL3, 18S, and EF1-α were found to be the top four choices for different tissues, whereas TUB was not found to favor qRT-PCR normalization in these tissues. Three stable reference genes are recommended for the normalization of qRT-PCR data in R. molle. Furthermore, the expression profiles of RmPSY (phytoene synthase) and RmPDS (phytoene dehydrogenase) were assessed using EF1-α, 18S, ACT5, RPL3, and their combination as internals. Similar trends were found, but these trends varied when the least stable reference gene TUB was used. The results further prove that it is necessary to validate the stability of reference genes prior to their use for normalization under different experimental conditions. This study provides useful information for reliable qRT-PCR data normalization in gene studies of R. molle. PMID:27803707

  15. Validation of internal reference genes for relative quantitation studies of gene expression in human laryngeal cancer

    PubMed Central

    Wang, Xiaofeng; He, Jinting; Wang, Wei; Ren, Ming; Gao, Sujie; Zhao, Guanjie

    2016-01-01

    Background The aim of this study was to determine the expression stabilities of 12 common internal reference genes for the relative quantitation analysis of target gene expression performed by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) in human laryngeal cancer. Methods Hep-2 cells and 14 laryngeal cancer tissue samples were investigated. The expression characteristics of 12 internal reference gene candidates (18S rRNA, GAPDH, ACTB, HPRT1, RPL29, HMBS, PPIA, ALAS1, TBP, PUM1, GUSB, and B2M) were assessed by RT-qPCR. The data were analyzed by three commonly used software programs: geNorm, NormFinder, and BestKeeper. Results The use of the combination of four internal reference genes was more appropriate than the use of a single internal reference gene. The optimal combination was PPIA + GUSB + RPL29 + HPRT1 for both the cell line and tissues; while the most appropriate combination was GUSB + RPL29 + HPRT1 + HMBS for the tissues. Conclusions Our recommended internal reference genes may improve the accuracy of relative quantitation analysis of target gene expression performed by the RT-qPCR method in further gene expression research on laryngeal tumors. PMID:27957397

  16. Cerebellin and des-cerebellin exert ACTH-like effects on corticosterone secretion and the intracellular signaling pathway gene expression in cultured rat adrenocortical cells--DNA microarray and QPCR studies.

    PubMed

    Rucinski, Marcin; Ziolkowska, Agnieszka; Szyszka, Marta; Malendowicz, Ludwik K

    2009-04-01

    Precerebellins (Cbln) belong to the C1q/TNF superfamily of secreted proteins which have diverse functions. They are abundantly expressed in the cerebellum, however, three of them are also expressed in the rat adrenal gland. All members of the Cbln family form homomeric and heteromeric complexes with each other in vitro and it was suggested that such complexes play a crucial role in normal development of the cerebellum. The aim of our study was to investigate whether Cbln1-derived peptides, cerebellin (CER) and des-Ser1-cerebellin (desCER) are involved in regulating biological functions of rat adrenocortical cells. In the primary culture of rat adrenocortical cells, 24 h exposure to CER or desCER notably stimulated corticosterone output and inhibited proliferative activity and similar effects were evoked by ACTH. To study gene transcript regulation by CER, desCER and ACTH, we applied Oligo GEArray DNA Microarray: Rat Signal Transduction Pathway Finder. In relation to the control culture, 13 of the 113 transcripts present on the array were differentially expressed. These transcripts were either up- or down-regulated by ACTH and/or CER or desCER treatment. Validation of DNA Microarray data by QPCR revealed that only 5 of 13 genes studied were differentially expressed. Of those genes, Fos and Icam1 were up-regulated and Egr1 was down-regulated by ACTH, CER and desCER. The remaining two genes, Fasn (insulin signaling pathway) and Hspb1 (HSP27) (stress signaling pathway), were regulated only by CER and desCER, but not by ACTH. Thus, both CER and desCER have effects similar to and different from corticotrophin on the intracellular signaling pathway gene expression in cultured rat adrenocortical cells.

  17. Identification of Reference Genes in Human Myelomonocytic Cells for Gene Expression Studies in Altered Gravity

    PubMed Central

    Thiel, Cora S.; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E.

    2015-01-01

    Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes (“housekeeping genes”) are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity. PMID:25654098

  18. Effect of ribonucleic acid (RNA) isolation methods on putative reference genes messenger RNA abundance in human spermatozoa.

    PubMed

    Barragán, M; Martínez, A; Llonch, S; Pujol, A; Vernaeve, V; Vassena, R

    2015-07-01

    Although the male gamete participates in a significant proportion of infertility cases, there are currently no proven molecular markers of sperm quality. The search for significant gene expression markers is partially hindered by the lack of a recognized set of reference genes (RGs) to normalize reverse transcription quantitative PCR (RT-qPCR) data across studies. The aim of this study is to define a set of RGs in assisted reproduction patients undergoing different sample collection and RNA isolation methods. Twenty-two normozoospermic men were included in the study. From each man, semen was either cryopreserved by slow freezing or analyzed fresh, and, for each, RNA was extracted with either phenol-free or phenol-based methods. In two cases, both methods were used to isolate RNA. Twenty putative RGs were analyzed and their mRNA abundance across samples was estimated by RT-qPCR. To determine the genes whose steady-state mRNA abundance remains unchanged, three different algorithms (geNorm, BestKeeper and NormFinder) were applied to the qPCR data. We found that RGs such as GAPDH or ACTB, useful in other biological contexts, cannot be used as reference for human spermatozoa. It is possible to compare gene expression from fresh and cryopreserved sperm samples using the same isolation method, while the mRNA abundance of expressed genes becomes different depending on the RNA isolation technique employed. In our conditions, the most appropriate RGs for RT-qPCR analysis were RPLP1, RPL13A, and RPLP2. Published discrepancies in gene expression studies in human spermatozoa may be due in part to inappropriate RGs selection, suggesting a possible different interpretation of PCR data in several reports, which were normalized using unstable RGs.

  19. Critical appraisal of quantitative PCR results in colorectal cancer research: can we rely on published qPCR results?

    PubMed

    Dijkstra, J R; van Kempen, L C; Nagtegaal, I D; Bustin, S A

    2014-06-01

    The use of real-time quantitative polymerase chain reaction (qPCR) in cancer research has become ubiquitous. The relative simplicity of qPCR experiments, which deliver fast and cost-effective results, means that each year an increasing number of papers utilizing this technique are being published. But how reliable are the published results? Since the validity of gene expression data is greatly dependent on appropriate normalisation to compensate for sample-to-sample and run-to-run variation, we have evaluated the adequacy of normalisation procedures in qPCR-based experiments. Consequently, we assessed all colorectal cancer publications that made use of qPCR from 2006 until August 2013 for the number of reference genes used and whether they had been validated. Using even these minimal evaluation criteria, the validity of only three percent (6/179) of the publications can be adequately assessed. We describe common errors, and conclude that the current state of reporting on qPCR in colorectal cancer research is disquieting. Extrapolated to the study of cancer in general, it is clear that the majority of studies using qPCR cannot be reliably assessed and that at best, the results of these studies may or may not be valid and at worst, pervasive incorrect normalisation is resulting in the wholesale publication of incorrect conclusions. This survey demonstrates that the existence of guidelines, such as MIQE, is necessary but not sufficient to address this problem and suggests that the scientific community should examine its responsibility and be aware of the implications of these findings for current and future research.

  20. Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces.

    PubMed

    Verhaegen, Bavo; De Reu, Koen; De Zutter, Lieven; Verstraete, Karen; Heyndrickx, Marc; Van Coillie, Els

    2016-05-18

    Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC) and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR), which has its specific limitations. Droplet digital PCR (ddPCR) has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014) for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan(®) Environmental Master Mix 2.0; UMM: TaqMan(®) Universal PCR Master Mix) were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material.

  1. Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces

    PubMed Central

    Verhaegen, Bavo; De Reu, Koen; De Zutter, Lieven; Verstraete, Karen; Heyndrickx, Marc; Van Coillie, Els

    2016-01-01

    Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC) and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR), which has its specific limitations. Droplet digital PCR (ddPCR) has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014) for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan® Environmental Master Mix 2.0; UMM: TaqMan® Universal PCR Master Mix) were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material. PMID:27213452

  2. Identifying suitable reference genes for gene expression analysis in developing skeletal muscle in pigs

    PubMed Central

    Zhang, YuanYuan; Hua, Chaoju; Wang, Zishuai; Li, Kui

    2016-01-01

    The selection of suitable reference genes is crucial to accurately evaluate and normalize the relative expression level of target genes for gene function analysis. However, commonly used reference genes have variable expression levels in developing skeletal muscle. There are few reports that systematically evaluate the expression stability of reference genes across prenatal and postnatal developing skeletal muscle in mammals. Here, we used quantitative PCR to examine the expression levels of 15 candidate reference genes (ACTB, GAPDH, RNF7, RHOA, RPS18, RPL32, PPIA, H3F3, API5, B2M, AP1S1, DRAP1, TBP, WSB, and VAPB) in porcine skeletal muscle at 26 different developmental stages (15 prenatal and 11 postnatal periods). We evaluated gene expression stability using the computer algorithms geNorm, NormFinder, and BestKeeper. Our results indicated that GAPDH and ACTB had the greatest variability among the candidate genes across prenatal and postnatal stages of skeletal muscle development. RPS18, API5, and VAPB had stable expression levels in prenatal stages, whereas API5, RPS18, RPL32, and H3F3 had stable expression levels in postnatal stages. API5 and H3F3 expression levels had the greatest stability in all tested prenatal and postnatal stages, and were the most appropriate reference genes for gene expression normalization in developing skeletal muscle. Our data provide valuable information for gene expression analysis during different stages of skeletal muscle development in mammals. This information can provide a valuable guide for the analysis of human diseases. PMID:27994956

  3. Identifying suitable reference genes for gene expression analysis in developing skeletal muscle in pigs.

    PubMed

    Niu, Guanglin; Yang, Yalan; Zhang, YuanYuan; Hua, Chaoju; Wang, Zishuai; Tang, Zhonglin; Li, Kui

    2016-01-01

    The selection of suitable reference genes is crucial to accurately evaluate and normalize the relative expression level of target genes for gene function analysis. However, commonly used reference genes have variable expression levels in developing skeletal muscle. There are few reports that systematically evaluate the expression stability of reference genes across prenatal and postnatal developing skeletal muscle in mammals. Here, we used quantitative PCR to examine the expression levels of 15 candidate reference genes (ACTB, GAPDH, RNF7, RHOA, RPS18, RPL32, PPIA, H3F3, API5, B2M, AP1S1, DRAP1, TBP, WSB, and VAPB) in porcine skeletal muscle at 26 different developmental stages (15 prenatal and 11 postnatal periods). We evaluated gene expression stability using the computer algorithms geNorm, NormFinder, and BestKeeper. Our results indicated that GAPDH and ACTB had the greatest variability among the candidate genes across prenatal and postnatal stages of skeletal muscle development. RPS18, API5, and VAPB had stable expression levels in prenatal stages, whereas API5, RPS18, RPL32, and H3F3 had stable expression levels in postnatal stages. API5 and H3F3 expression levels had the greatest stability in all tested prenatal and postnatal stages, and were the most appropriate reference genes for gene expression normalization in developing skeletal muscle. Our data provide valuable information for gene expression analysis during different stages of skeletal muscle development in mammals. This information can provide a valuable guide for the analysis of human diseases.

  4. Validation of commonly used reference genes for sleep-related gene expression studies

    PubMed Central

    Lee, Kil S; Alvarenga, Tathiana A; Guindalini, Camila; Andersen, Monica L; Castro, Rosa MRPS; Tufik, Sergio

    2009-01-01

    Background Sleep is a restorative process and is essential for maintenance of mental and physical health. In an attempt to understand the complexity of sleep, multidisciplinary strategies, including genetic approaches, have been applied to sleep research. Although quantitative real time PCR has been used in previous sleep-related gene expression studies, proper validation of reference genes is currently lacking. Thus, we examined the effect of total or paradoxical sleep deprivation (TSD or PSD) on the expression stability of the following frequently used reference genes in brain and blood: beta-actin (b-actin), beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and hypoxanthine guanine phosphoribosyl transferase (HPRT). Results Neither TSD nor PSD affected the expression stability of all tested genes in both tissues indicating that b-actin, B2M, GAPDH and HPRT are appropriate reference genes for the sleep-related gene expression studies. In order to further verify these results, the relative expression of brain derived neurotrophic factor (BDNF) and glycerol-3-phosphate dehydrogenase1 (GPD1) was evaluated in brain and blood, respectively. The normalization with each of four reference genes produced similar pattern of expression in control and sleep deprived rats, but subtle differences in the magnitude of expression fold change were observed which might affect the statistical significance. Conclusion This study demonstrated that sleep deprivation does not alter the expression stability of commonly used reference genes in brain and blood. Nonetheless, the use of multiple reference genes in quantitative RT-PCR is required for the accurate results. PMID:19445681

  5. Using nonlinear least squares to assess relative expression and its uncertainty in real-time qPCR studies.

    PubMed

    Tellinghuisen, Joel

    2016-03-01

    Relative expression ratios are commonly estimated in real-time qPCR studies by comparing the quantification cycle for the target gene with that for a reference gene in the treatment samples, normalized to the same quantities determined for a control sample. For the "standard curve" design, where data are obtained for all four of these at several dilutions, nonlinear least squares can be used to assess the amplification efficiencies (AE) and the adjusted ΔΔCq and its uncertainty, with automatic inclusion of the effect of uncertainty in the AEs. An algorithm is illustrated for the KaleidaGraph program.

  6. KEGG as a reference resource for gene and protein annotation

    PubMed Central

    Kanehisa, Minoru; Sato, Yoko; Kawashima, Masayuki; Furumichi, Miho; Tanabe, Mao

    2016-01-01

    KEGG (http://www.kegg.jp/ or http://www.genome.jp/kegg/) is an integrated database resource for biological interpretation of genome sequences and other high-throughput data. Molecular functions of genes and proteins are associated with ortholog groups and stored in the KEGG Orthology (KO) database. The KEGG pathway maps, BRITE hierarchies and KEGG modules are developed as networks of KO nodes, representing high-level functions of the cell and the organism. Currently, more than 4000 complete genomes are annotated with KOs in the KEGG GENES database, which can be used as a reference data set for KO assignment and subsequent reconstruction of KEGG pathways and other molecular networks. As an annotation resource, the following improvements have been made. First, each KO record is re-examined and associated with protein sequence data used in experiments of functional characterization. Second, the GENES database now includes viruses, plasmids, and the addendum category for functionally characterized proteins that are not represented in complete genomes. Third, new automatic annotation servers, BlastKOALA and GhostKOALA, are made available utilizing the non-redundant pangenome data set generated from the GENES database. As a resource for translational bioinformatics, various data sets are created for antimicrobial resistance and drug interaction networks. PMID:26476454

  7. Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions.

    PubMed

    Chen, Lei; Zhong, Hai-ying; Kuang, Jian-fei; Li, Jian-guo; Lu, Wang-jin; Chen, Jian-ye

    2011-08-01

    Reverse transcription quantitative real-time PCR (RT-qPCR) is a sensitive technique for quantifying gene expression, but its success depends on the stability of the reference gene(s) used for data normalization. Only a few studies on validation of reference genes have been conducted in fruit trees and none in banana yet. In the present work, 20 candidate reference genes were selected, and their expression stability in 144 banana samples were evaluated and analyzed using two algorithms, geNorm and NormFinder. The samples consisted of eight sample sets collected under different experimental conditions, including various tissues, developmental stages, postharvest ripening, stresses (chilling, high temperature, and pathogen), and hormone treatments. Our results showed that different suitable reference gene(s) or combination of reference genes for normalization should be selected depending on the experimental conditions. The RPS2 and UBQ2 genes were validated as the most suitable reference genes across all tested samples. More importantly, our data further showed that the widely used reference genes, ACT and GAPDH, were not the most suitable reference genes in many banana sample sets. In addition, the expression of MaEBF1, a gene of interest that plays an important role in regulating fruit ripening, under different experimental conditions was used to further confirm the validated reference genes. Taken together, our results provide guidelines for reference gene(s) selection under different experimental conditions and a foundation for more accurate and widespread use of RT-qPCR in banana.

  8. Reference gene alternatives to Gapdh in rodent and human heart failure gene expression studies

    PubMed Central

    2010-01-01

    Background Quantitative real-time RT-PCR (RT-qPCR) is a highly sensitive method for mRNA quantification, but requires invariant expression of the chosen reference gene(s). In pathological myocardium, there is limited information on suitable reference genes other than the commonly used Gapdh mRNA and 18S ribosomal RNA. Our aim was to evaluate and identify suitable reference genes in human failing myocardium, in rat and mouse post-myocardial infarction (post-MI) heart failure and across developmental stages in fetal and neonatal rat myocardium. Results The abundance of Arbp, Rpl32, Rpl4, Tbp, Polr2a, Hprt1, Pgk1, Ppia and Gapdh mRNA and 18S ribosomal RNA in myocardial samples was quantified by RT-qPCR. The expression variability of these transcripts was evaluated by the geNorm and Normfinder algorithms and by a variance component analysis method. Biological variability was a greater contributor to sample variability than either repeated reverse transcription or PCR reactions. Conclusions The most stable reference genes were Rpl32, Gapdh and Polr2a in mouse post-infarction heart failure, Polr2a, Rpl32 and Tbp in rat post-infarction heart failure and Rpl32 and Pgk1 in human heart failure (ischemic disease and cardiomyopathy). The overall most stable reference genes across all three species was Rpl32 and Polr2a. In rat myocardium, all reference genes tested showed substantial variation with developmental stage, with Rpl4 as was most stable among the tested genes. PMID:20331858

  9. Validation of internal reference genes for quantitative real-time PCR in a non-model organism, the yellow-necked mouse, Apodemus flavicollis

    PubMed Central

    2009-01-01

    Background Reference genes are used as internal standards to normalize mRNA abundance in quantitative real-time PCR and thereby allow a direct comparison between samples. So far most of these expression studies used human or classical laboratory model species whereas studies on non-model organism under in-situ conditions are quite rare. However, only studies in free-ranging populations can reveal the effects of natural selection on the expression levels of functional important genes. In order to test the feasibility of gene expression studies in wildlife samples we transferred and validated potential reference genes that were developed for lab mice (Mus musculus) to samples of wild yellow-necked mice, Apodemus flavicollis. The stability and suitability of eight potential reference genes was accessed by the programs BestKeeper, NormFinder and geNorm. Findings Although the three programs used different algorithms the ranking order of reference genes was significantly concordant and geNorm differed in only one, NormFinder in two positions compared to BestKeeper. The genes ordered by their mean rank from the most to the least stable gene were: Rps18, Sdha, Canx, Actg1, Pgk1, Ubc, Rpl13a and Actb. Analyses of the normalization factor revealed best results when the five most stable genes were included for normalization. Discussion We established a SYBR green qPCR assay for liver samples of wild A. flavicollis and conclude that five genes should be used for appropriate normalization. Our study provides the basis to investigate differential expression of genes under selection under natural selection conditions in liver samples of A. flavicollis. This approach might also be applicable to other non-model organisms. PMID:20030847

  10. Assessing mycoplasma contamination of cell cultures by qPCR using a set of universal primer pairs targeting a 1.5 kb fragment of 16S rRNA genes

    PubMed Central

    Jean, Audrey; Tardy, Florence; Allatif, Omran; Grosjean, Isabelle; Blanquier, Bariza

    2017-01-01

    Mycoplasmas (a generic name for Mollicutes) are a predominant bacterial contaminant of cell culture and cell derived products including viruses. This prokaryote class is characterized by very small size and lack of a cell wall. Consequently, mycoplasmas escape ultrafiltration and visualization under routine microscopic examination, hence the ease with which cells in culture can be contaminated, with routinely more than 10% of cell lines being contaminated. Mycoplasma are a formidable threat both in fundamental research by perverting a whole range of cell properties and functions and in the pharmacological use of cells and cell derived products. Although many methods have been developed, there is still a need for a sensitive, universal assay. Here is reported the development and validation of a quantitative polymerase chain reaction (qPCR) based on the amplification of a 1.5 kb fragment covering the 16S rDNA of the Mollicute class by real-time PCR using universal U1 and U8 degenerate primers. The method includes the addition of a DNA loading probe to each sample to monitor DNA extraction and the absence of PCR inhibitors in the extracted DNA, a positive mycoplasma 16S rDNA traceable reference sample to exclude any accidental contamination of an unknown sample with this reference DNA, an analysis procedure based on the examination of the melting curve and the size of the PCR amplicon, followed by quantification of the number of 16S rDNA copies (with a lower limit of 19 copies) when relevant, and, if useful, the identification of the contaminating prokaryote by sequencing. The method was validated on a collection of mycoplasma strains and by testing over 100 samples of unknown contamination status including stocks of viruses requiring biosafety level 2, 3 or 4 containments. When compared to four established methods, the m16S_qPCR technique exhibits the highest sensitivity in detecting mycoplasma contamination. PMID:28225826

  11. Careful Selection of Reference Genes Is Required for Reliable Performance of RT-qPCR in Human Normal and Cancer Cell Lines

    PubMed Central

    Jacob, Francis; Guertler, Rea; Naim, Stephanie; Nixdorf, Sheri; Fedier, André; Hacker, Neville F.; Heinzelmann-Schwarz, Viola

    2013-01-01

    Reverse Transcription - quantitative Polymerase Chain Reaction (RT-qPCR) is a standard technique in most laboratories. The selection of reference genes is essential for data normalization and the selection of suitable reference genes remains critical. Our aim was to 1) review the literature since implementation of the MIQE guidelines in order to identify the degree of acceptance; 2) compare various algorithms in their expression stability; 3) identify a set of suitable and most reliable reference genes for a variety of human cancer cell lines. A PubMed database review was performed and publications since 2009 were selected. Twelve putative reference genes were profiled in normal and various cancer cell lines (n = 25) using 2-step RT-qPCR. Investigated reference genes were ranked according to their expression stability by five algorithms (geNorm, Normfinder, BestKeeper, comparative ΔCt, and RefFinder). Our review revealed 37 publications, with two thirds patient samples and one third cell lines. qPCR efficiency was given in 68.4% of all publications, but only 28.9% of all studies provided RNA/cDNA amount and standard curves. GeNorm and Normfinder algorithms were used in 60.5% in combination. In our selection of 25 cancer cell lines, we identified HSPCB, RRN18S, and RPS13 as the most stable expressed reference genes. In the subset of ovarian cancer cell lines, the reference genes were PPIA, RPS13 and SDHA, clearly demonstrating the necessity to select genes depending on the research focus. Moreover, a cohort of at least three suitable reference genes needs to be established in advance to the experiments, according to the guidelines. For establishing a set of reference genes for gene normalization we recommend the use of ideally three reference genes selected by at least three stability algorithms. The unfortunate lack of compliance to the MIQE guidelines reflects that these need to be further established in the research community. PMID:23554992

  12. Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells.

    PubMed

    Cieslak, Jakub; Mackowski, Mariusz; Czyzak-Runowska, Grazyna; Wojtowski, Jacek; Puppel, Kamila; Kuczynska, Beata; Pawlak, Piotr

    2015-01-01

    Apart from the well-known role of somatic cell count as a parameter reflecting the inflammatory status of the mammary gland, the composition of cells isolated from milk is considered as a valuable material for gene expression studies in mammals. Due to its unique composition, in recent years an increasing interest in mare's milk consumption has been observed. Thus, investigating the genetic background of horse's milk variability presents and interesting study model. Relying on 39 milk samples collected from mares representing three breeds (Polish Primitive Horse, Polish Cold-blooded Horse, Polish Warmblood Horse) we aimed to investigate the utility of equine milk somatic cells as a source of mRNA and to screen the best reference genes for RT-qPCR using geNorm and NormFinder algorithms. The results showed that despite relatively low somatic cell counts in mare's milk, the amount and the quality of the extracted RNA are sufficient for gene expression studies. The analysis of the utility of 7 potential reference genes for RT-qPCR experiments for the normalization of equine milk somatic cells revealed some differences between the outcomes of the applied algorithms, although in both cases the KRT8 and TOP2B genes were pointed as the most stable. Analysis by geNorm showed that the combination of 4 reference genes (ACTB, GAPDH, TOP2B and KRT8) is required for apropriate RT-qPCR experiments normalization, whereas NormFinder algorithm pointed the combination of KRT8 and RPS9 genes as the most suitable. The trial study of the relative transcript abundance of the beta-casein gene with the use of various types and numbers of internal control genes confirmed once again that the selection of proper reference gene combinations is crucial for the final results of each real-time PCR experiment.

  13. Selection and Validation of Reference Genes for qRT-PCR in Cycas elongata

    PubMed Central

    Deng, Tian; Chen, Letian; Wu, Hong; Zhang, Shouzhou

    2016-01-01

    Quantitative reverse transcription PCR (qRT-PCR) is a sensitive technique used in gene expression studies. To achieve a reliable quantification of transcripts, appropriate reference genes are required for comparison of transcripts in different samples. However, few reference genes are available for non-model taxa, and to date, reliable reference genes in Cycas elongata have not been well characterized. In this study, 13 reference genes (ACT7, TUB, UBQ, EIF4, EF1, CLATHRIN1, PP2A, RPB2, GAPC2, TIP41, MAPK, SAMDC and CYP) were chosen from the transcriptome database of C. elongata, and these genes were evaluated in 8 different organ samples. Three software programs, NormFinder, GeNorm and BestKeeper, were used to validate the stability of the potential reference genes. Results obtained from these three programs suggested that CeGAPC2 and CeRPB2 are the most stable reference genes, while CeACT7 is the least stable one among the 13 tested genes. Further confirmation of the identified reference genes was established by the relative expression of AGAMOUSE gene of C. elongata (CeAG). While our stable reference genes generated consistent expression patterns in eight tissues, we note that our results indicate that an inappropriate reference gene might cause erroneous results. Our systematic analysis for stable reference genes of C. elongata facilitates further gene expression studies and functional analyses of this species. PMID:27124298

  14. Investigation of Reference Genes in Vibrio parahaemolyticus for Gene Expression Analysis Using Quantitative RT-PCR

    PubMed Central

    Ma, Yue-jiao; Sun, Xiao-hong; Xu, Xiao-yan; Zhao, Yong; Pan, Ying-jie; Hwang, Cheng-An; Wu, Vivian C. H.

    2015-01-01

    Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize its virulence factors and understand the effect of environmental conditions on its pathogenicity. However, there is not a stable gene in V. parahaemolyticus that has been identified for use as a reference gene for qRT-PCR. This study evaluated the stability of 6 reference genes (16S rRNA, recA, rpoS, pvsA, pvuA, and gapdh) in 5 V. parahaemolyticus strains (O3:K6-clinical strain-tdh+, ATCC33846-tdh+, ATCC33847-tdh+, ATCC17802-trh+, and F13-environmental strain-tdh+) cultured at 4 different temperatures (15, 25, 37 and 42°C). Stability values were calculated using GeNorm, NormFinder, BestKeeper, and Delta CT algorithms. The results indicated that recA was the most stably expressed gene in the V. parahaemolyticus strains cultured at different temperatures. This study examined multiple V. parahaemolyticus strains and growth temperatures, hence the finding provided stronger evidence that recA can be used as a reference gene for gene expression studies in V. parahaemolyticus. PMID:26659406

  15. Evaluation and selection of reliable reference genes for gene expression under abiotic stress in cotton (Gossypium hirsutum L.).

    PubMed

    Wang, Min; Wang, Qinglian; Zhang, Baohong

    2013-11-01

    Reference genes are critical for normalization of the gene expression level of target genes. The widely used housekeeping genes may change their expression levels at different tissue under different treatment or stress conditions. Therefore, systematical evaluation on the housekeeping genes is required for gene expression analysis. Up to date, no work was performed to evaluate the housekeeping genes in cotton under stress treatment. In this study, we chose 10 housekeeping genes to systematically assess their expression levels at two different tissues (leaves and roots) under two different abiotic stresses (salt and drought) with three different concentrations. Our results show that there is no best reference gene for all tissues at all stress conditions. The reliable reference gene should be selected based on a specific condition. For example, under salt stress, UBQ7, GAPDH and EF1A8 are better reference genes in leaves; TUA10, UBQ7, CYP1, GAPDH and EF1A8 were better in roots. Under drought stress, UBQ7, EF1A8, TUA10, and GAPDH showed less variety of expression level in leaves and roots. Thus, it is better to identify reliable reference genes first before performing any gene expression analysis. However, using a combination of housekeeping genes as reference gene may provide a new strategy for normalization of gene expression. In this study, we found that combination of four housekeeping genes worked well as reference genes under all the stress conditions.

  16. Evaluation of RNA extraction methods and identification of putative reference genes for real-time quantitative polymerase chain reaction expression studies on olive (Olea europaea L.) fruits.

    PubMed

    Nonis, Alberto; Vezzaro, Alice; Ruperti, Benedetto

    2012-07-11

    Genome wide transcriptomic surveys together with targeted molecular studies are uncovering an ever increasing number of differentially expressed genes in relation to agriculturally relevant processes in olive (Olea europaea L). These data need to be supported by quantitative approaches enabling the precise estimation of transcript abundance. qPCR being the most widely adopted technique for mRNA quantification, preliminary work needs to be done to set up robust methods for extraction of fully functional RNA and for the identification of the best reference genes to obtain reliable quantification of transcripts. In this work, we have assessed different methods for their suitability for RNA extraction from olive fruits and leaves and we have evaluated thirteen potential candidate reference genes on 21 RNA samples belonging to fruit developmental/ripening series and to leaves subjected to wounding. By using two different algorithms, GAPDH2 and PP2A1 were identified as the best reference genes for olive fruit development and ripening, and their effectiveness for normalization of expression of two ripening marker genes was demonstrated.

  17. Easy-to-use strategy for reference gene selection in quantitative real-time PCR experiments.

    PubMed

    Klenke, Stefanie; Renckhoff, Kristina; Engler, Andrea; Peters, Jürgen; Frey, Ulrich H

    2016-12-01

    Real-time PCR is an indispensable technique for mRNA expression analysis but conclusions depend on appropriate reference gene selection. However, while reference gene selection has been a topic of publications, this issue is often disregarded when measuring target mRNA expression. Therefore, we (1) evaluated the frequency of appropriate reference gene selection, (2) suggest an easy-to-use tool for least variability reference gene selection, (3) demonstrate application of this tool, and (4) show effects on target gene expression profiles. All 2015 published articles in Naunyn-Schmiedeberg's Archives of Pharmacology were screened for the use of quantitative real-time PCR analysis and selection of reference genes. Target gene expression (Vegfa, Grk2, Sirt4, and Timp3) in H9c2 cells was analyzed following various interventions (hypoxia, hyperglycemia, and/or isoflurane exposure with and without subsequent hypoxia) in relation to putative reference genes (Actb, Gapdh, B2m, Sdha, and Rplp1) using the least variability method vs. an arbitrarily selected but established reference gene. In the vast majority (18 of 21) of papers, no information was provided regarding selection of an appropriate reference gene. In only 1 of 21 papers, a method of appropriate reference gene selection was described and in 2 papers reference gene selection remains unclear. The method of reference gene selection had major impact on interpretation of target gene expression. With hypoxia, for instance, the least variability gene was Rplp1 and target gene expression (Vefga) heavily showed a 2-fold up-regulation (p = 0.022) but no change (p = 0.3) when arbitrarily using Gapdh. Frequency of appropriate reference gene selection in this journal is low, and we propose our strategy for reference gene selection as an easy tool for proper target gene expression.

  18. Pneumocystis sp. in bats evaluated by qPCR.

    PubMed

    Cavallini Sanches, E M; Ferreiro, L; Andrade, C P; Pacheco, S M; Almeida, L L; Spanamberg, A; Wissmann, G

    2013-03-01

    Molecular techniques have revealed a high prevalence of Pneumocystis colonization in wild mammals. Accurate quantification of Pneumocystis sp. is essential for the correct interpretation of many research experiments investigating this organism. The objectives of this study were to detect the presence of Pneumocystis sp. in bats by qPCR, and to distinguish colonization from infection. Probes and primers for real time PCR (qPCR) were designed based on the gene of major surface glycoprotein (MSG) of Pneumocystis sp., in order to analyze 195 lung tissue samples from bats captured (2007-2009). All samples were also analyzed by nested PCR, using oligonucleotide primers designed for the gene encoding the mitochondrial small subunit rRNA (mtSSU rRNA) to confirm the results. The qPCR assay was standardized using a standard curve made with the DNA extracted from bronchoalveolar lavage positive for Pneumocystis jirovecii. The average Ct was found to be between 13 and 14 (calibration curve) for the detection of infection with Pneumocystis sp. and above these values for colonization. It was considered as negative samples the ones that had Ct values equal to 50. Out of the total 195 samples, 47 (24.1%) bat lung DNA samples were positive for Pneumocystis sp. by qPCR. The most common bat species found were: Tadarida brasiliensis (23.4%), Histiotus velatus (17.0%), Desmodus rotundus (14.9%) and Molossus molossus (8.5%). The average cycle threshold of the positive samples (bats) was 25.8 and standard deviation was 1.7. The DNA samples with Ct values greater than 14 suggest that these animals might be colonized by Pneumocystis sp. Results obtained in this study demonstrated the usefulness of the qPCR procedure for identification of Pneumocystis sp. and for distinction between its colonizing or infectious status in bats.

  19. Identification of suitable reference genes for gene expression normalization in qRT-PCR analysis in watermelon.

    PubMed

    Kong, Qiusheng; Yuan, Jingxian; Gao, Lingyun; Zhao, Shuang; Jiang, Wei; Huang, Yuan; Bie, Zhilong

    2014-01-01

    Watermelon is one of the major Cucurbitaceae crops and the recent availability of genome sequence greatly facilitates the fundamental researches on it. Quantitative real-time reverse transcriptase PCR (qRT-PCR) is the preferred method for gene expression analyses, and using validated reference genes for normalization is crucial to ensure the accuracy of this method. However, a systematic validation of reference genes has not been conducted on watermelon. In this study, transcripts of 15 candidate reference genes were quantified in watermelon using qRT-PCR, and the stability of these genes was compared using geNorm and NormFinder. geNorm identified ClTUA and ClACT, ClEF1α and ClACT, and ClCAC and ClTUA as the best pairs of reference genes in watermelon organs and tissues under normal growth conditions, abiotic stress, and biotic stress, respectively. NormFinder identified ClYLS8, ClUBCP, and ClCAC as the best single reference genes under the above experimental conditions, respectively. ClYLS8 and ClPP2A were identified as the best reference genes across all samples. Two to nine reference genes were required for more reliable normalization depending on the experimental conditions. The widely used watermelon reference gene 18SrRNA was less stable than the other reference genes under the experimental conditions. Catalase family genes were identified in watermelon genome, and used to validate the reliability of the identified reference genes. ClCAT1and ClCAT2 were induced and upregulated in the first 24 h, whereas ClCAT3 was downregulated in the leaves under low temperature stress. However, the expression levels of these genes were significantly overestimated and misinterpreted when 18SrRNA was used as a reference gene. These results provide a good starting point for reference gene selection in qRT-PCR analyses involving watermelon.

  20. Automatic extraction of reference gene from literature in plants based on texting mining.

    PubMed

    He, Lin; Shen, Gengyu; Li, Fei; Huang, Shuiqing

    2015-01-01

    Real-Time Quantitative Polymerase Chain Reaction (qRT-PCR) is widely used in biological research. It is a key to the availability of qRT-PCR experiment to select a stable reference gene. However, selecting an appropriate reference gene usually requires strict biological experiment for verification with high cost in the process of selection. Scientific literatures have accumulated a lot of achievements on the selection of reference gene. Therefore, mining reference genes under specific experiment environments from literatures can provide quite reliable reference genes for similar qRT-PCR experiments with the advantages of reliability, economic and efficiency. An auxiliary reference gene discovery method from literature is proposed in this paper which integrated machine learning, natural language processing and text mining approaches. The validity tests showed that this new method has a better precision and recall on the extraction of reference genes and their environments.

  1. Validation of reference genes for quantitative gene expression studies in Volvox carteri using real-time RT-PCR.

    PubMed

    Kianianmomeni, Arash; Hallmann, Armin

    2013-12-01

    Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is a sensitive technique for analysis of gene expression under a wide diversity of biological conditions. However, the identification of suitable reference genes is a critical factor for analysis of gene expression data. To determine potential reference genes for normalization of qRT-PCR data in the green alga Volvox carteri, the transcript levels of ten candidate reference genes were measured by qRT-PCR in three experimental sample pools containing different developmental stages, cell types and stress treatments. The expression stability of the candidate reference genes was then calculated using the algorithms geNorm, NormFinder and BestKeeper. The genes for 18S ribosomal RNA (18S) and eukaryotic translation elongation factor 1α2 (eef1) turned out to have the most stable expression levels among the samples both from different developmental stages and different stress treatments. The genes for the ribosomal protein L23 (rpl23) and the TATA-box binding protein (tbpA) showed equivalent transcript levels in the comparison of different cell types, and therefore, can be used as reference genes for cell-type specific gene expression analysis. Our results indicate that more than one reference gene is required for accurate normalization of qRT-PCRs in V. carteri. The reference genes in our study show a much better performance than the housekeeping genes used as a reference in previous studies.

  2. Candidate qRT-PCR reference genes for barley that demonstrate better stability than traditional housekeeping genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gene transcript expression analysis is a useful tool for correlating gene activity with plant phenotype. For these studies, an appropriate reference gene is necessary to quantify the expression of target genes. Classic housekeeping genes have often been used for this purpose, but may not be consis...

  3. Validation of Suitable Reference Genes for Assessing Gene Expression of MicroRNAs in Lonicera japonica

    PubMed Central

    Wang, Yaolong; Liu, Juan; Wang, Xumin; Liu, Shuang; Wang, Guoliang; Zhou, Junhui; Yuan, Yuan; Chen, Tiying; Jiang, Chao; Zha, Liangping; Huang, Luqi

    2016-01-01

    MicroRNAs (miRNAs), which play crucial regulatory roles in plant secondary metabolism and responses to the environment, could be developed as promising biomarkers for different varieties and production areas of herbal medicines. However, limited information is available for miRNAs from Lonicera japonica, which is widely used in East Asian countries owing to various pharmaceutically active secondary metabolites. Selection of suitable reference genes for quantification of target miRNA expression through quantitative real-time (qRT)-PCR is important for elucidating the molecular mechanisms of secondary metabolic regulation in different tissues and varieties of L. japonica. For precise normalization of gene expression data in L. japonica, 16 candidate miRNAs were examined in three tissues, as well as 21 cultivated varieties collected from 16 production areas, using GeNorm, NormFinder, and RefFinder algorithms. Our results revealed combination of u534122 and u3868172 as the best reference genes across all samples. Their specificity was confirmed by detecting the cycling threshold (Ct) value ranges in different varieties of L. japonica collected from diverse production areas, suggesting the use of these two reference miRNAs is sufficient for accurate transcript normalization with different tissues, varieties, and production areas. To our knowledge, this is the first report on validation of reference miRNAs in honeysuckle (Lonicera spp.). Restuls from this study can further facilitate discovery of functional regulatory miRNAs in different varieties of L. japonica. PMID:27507983

  4. Identification of suitable reference genes for investigating gene expression in human gallbladder carcinoma using reverse transcription quantitative polymerase chain reaction.

    PubMed

    Yu, Shan; Yang, Qiwei; Yang, Jing Hui; Du, Zhenwu; Zhang, Guizhen

    2015-04-01

    Reverse transcription quantitative polymerase chain reaction (RT‑qPCR) has become a frequently used strategy in gene expression studies. The relative quantification method is an important and commonly used method for the evaluation of RT‑qPCR data. The key aim of this method is to identify an applicable internal reference gene, however, there are currently no suitable reference genes for gene analysis in gallbladder carcinoma. In the present study, screening was performed using 12 common reference genes, which were selected in order to provide an experimental basis for the investigation of gene expression in gallbladder carcinoma. A total of 16 tissue samples of gallbladder carcinoma and their matched normal gallbladder tissues were used. The gene expression stability and applicability of the 12 reference gene candidates were determined using the geNorm, NormFinder and BestKeeper software programs. Following comparison of the results of the three software programs, HPRT1 was identified as the most stably expressed reference gene. In the normal gallbladder group, the relative stably expressed reference gene was PPIA and in the entire sample group, the relatively stably expressed reference gene was PPIA. The present study also demonstrated that the combination of the three reference genes was the most appropriate. The recommended combinations were PPIA + PUM1 + ACTB for the total sample group, GAPDH + PBGD + ALAS1 for the gallbladder carcinoma group and PPIA + PUM1 + TBP for the paired normal gallbladder group.

  5. Identification of suitable reference genes for gene expression studies by qRT-PCR in the blister beetle Mylabris cichorii.

    PubMed

    Wang, Yu; Wang, Zhong-Kang; Huang, Yi; Liao, Yu-Feng; Yin, You-Ping

    2014-01-01

    The blister beetle Mylabris cichorii L. (Coleoptera: Meloidae) is a traditional medicinal insect recorded in the Chinese Pharmacopoeia. It synthesizes cantharidin, which kills cancer cells efficiently. Only males produce large amounts of cantharidin. Reference genes are required as endogenous controls for the analysis of differential gene expression in M. cichorii. Our study chose 10 genes as candidate reference genes. The stability of expression of these genes was analyzed by quantitative PCR and determined with two algorithms, geNorm and Normfinder. We recommend UBE3A and RPL22e as suitable reference genes in females and UBE3A, TAF5, and RPL22e in males.

  6. Identification of Suitable Reference Genes for gene Expression Studies by qRT-PCR in the Blister Beetle Mylabris cichorii

    PubMed Central

    Wang, Yu; Wang, Zhong-Kang; Huang, Yi; Liao, Yu-Feng; Yin, You-Ping

    2014-01-01

    The blister beetle Mylabris cichorii L. (Coleoptera: Meloidae) is a traditional medicinal insect recorded in the Chinese Pharmacopoeia. It synthesizes cantharidin, which kills cancer cells efficiently. Only males produce large amounts of cantharidin. Reference genes are required as endogenous controls for the analysis of differential gene expression in M. cichorii. Our study chose 10 genes as candidate reference genes. The stability of expression of these genes was analyzed by quantitative PCR and determined with two algorithms, geNorm and Normfinder. We recommend UBE3A and RPL22e as suitable reference genes in females and UBE3A, TAF5, and RPL22e in males. PMID:25368050

  7. Identification and Validation of Reference Genes for qRT-PCR Studies of Gene Expression in Dioscorea opposita

    PubMed Central

    Zhao, Xiting; Zhang, Xiaoli; Guo, Xiaobo; Li, Shujie; Han, Linlin; Song, Zhihui; Wang, Yunying; Li, Junhua; Li, Mingjun

    2016-01-01

    Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most common methods for gene expression studies. Data normalization based on reference genes is essential for obtaining reliable results for qRT-PCR assays. This study evaluated potential reference genes of Chinese yam (Dioscorea opposita Thunb.), which is an important tuber crop and medicinal plant in East Asia. The expression of ten candidate reference genes across 20 samples from different organs and development stages was assessed. We identified the most stable genes for qRT-PCR studies using combined samples from different organs. Our results also suggest that different suitable reference genes or combinations of reference genes for normalization should be applied according to different organs and developmental stages. To validate the suitability of the reference genes, we evaluated the relative expression of PE2.1 and PE53, which are two genes that may be associated with microtuber formation. Our results provide the foundation for reference gene(s) selection in D. opposita and will contribute toward more accurate gene analysis studies of the genus Dioscorea. PMID:27314014

  8. Identification and Validation of Reference Genes for qRT-PCR Studies of Gene Expression in Dioscorea opposita.

    PubMed

    Zhao, Xiting; Zhang, Xiaoli; Guo, Xiaobo; Li, Shujie; Han, Linlin; Song, Zhihui; Wang, Yunying; Li, Junhua; Li, Mingjun

    2016-01-01

    Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most common methods for gene expression studies. Data normalization based on reference genes is essential for obtaining reliable results for qRT-PCR assays. This study evaluated potential reference genes of Chinese yam (Dioscorea opposita Thunb.), which is an important tuber crop and medicinal plant in East Asia. The expression of ten candidate reference genes across 20 samples from different organs and development stages was assessed. We identified the most stable genes for qRT-PCR studies using combined samples from different organs. Our results also suggest that different suitable reference genes or combinations of reference genes for normalization should be applied according to different organs and developmental stages. To validate the suitability of the reference genes, we evaluated the relative expression of PE2.1 and PE53, which are two genes that may be associated with microtuber formation. Our results provide the foundation for reference gene(s) selection in D. opposita and will contribute toward more accurate gene analysis studies of the genus Dioscorea.

  9. Genome-wide selection of superior reference genes for expression studies in Ganoderma lucidum.

    PubMed

    Xu, Zhichao; Xu, Jiang; Ji, Aijia; Zhu, Yingjie; Zhang, Xin; Hu, Yuanlei; Song, Jingyuan; Chen, Shilin

    2015-12-15

    Quantitative real-time polymerase chain reaction (qRT-PCR) is widely used for the accurate analysis of gene expression. However, high homology among gene families might result in unsuitability of reference genes, which leads to the inaccuracy of qRT-PCR analysis. The release of the Ganoderma lucidum genome has triggered numerous studies to be done on the homology among gene families with the purpose of selecting reliable reference genes. Based on the G. lucdum genome and transcriptome database, 38 candidate reference genes including 28 novel genes were systematically selected and evaluated for qRT-PCR normalization. The result indicated that commonly used polyubiquitin (PUB), beta-actin (BAT), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were unsuitable reference genes because of the high sequence similarity and low primer specificity. According to the evaluation of RefFinder, cyclophilin 5 (CYP5) was ranked as the most stable reference gene for 27 tested samples under all experimental conditions and eighteen mycelial samples. Based on sequence analysis and expression analysis, our study suggested that gene characteristic, primer specificity of high homologous genes, allele-specificity expression of candidate genes and under-evaluation of reference genes influenced the accuracy and sensitivity of qRT-PCR analysis. This investigation not only revealed potential factors influencing the unsuitability of reference genes but also selected the superior reference genes from more candidate genes and testing samples than those used in the previous study. Furthermore, our study established a model for reference gene analysis by using the genomic sequence.

  10. Identification and testing of reference genes for Sesame gene expression analysis by quantitative real-time PCR.

    PubMed

    Wei, Libin; Miao, Hongmei; Zhao, Ruihong; Han, Xiuhua; Zhang, Tide; Zhang, Haiyang

    2013-03-01

    Sesame (Sesamum indicum L.) is an ancient and important oilseed crop. However, few sesame reference genes have been selected for quantitative real-time PCR until now. Screening and validating reference genes is a requisite for gene expression normalization in sesame functional genomics research. In this study, ten candidate reference genes, i.e., SiACT, SiUBQ6, SiTUB, Si18S rRNA, SiEF1α, SiCYP, SiHistone, SiDNAJ, SiAPT and SiGAPDH, were chosen and examined systematically in 32 sesame samples. Three qRT-PCR analysis methods, i.e., geNorm, NormFinder and BestKeeper, were evaluated systematically. Results indicated that all ten candidate reference genes could be used as reference genes in sesame. SiUBQ6 and SiAPT were the optimal reference genes for sesame plant development; SiTUB was suitable for sesame vegetative tissue development, SiDNAJ for pathogen treatment, SiHistone for abiotic stress, SiUBQ6 for bud development and SiACT for seed germination. As for hormone treatment and seed development, SiHistone, SiCYP, SiDNAJ or SiUBQ6, as well as SiACT, SiDNAJ, SiTUB or SiAPT, could be used as reference gene, respectively. To illustrate the suitability of these reference genes, we analyzed the expression variation of three functional sesame genes of SiSS, SiLEA and SiGH in different organs using the optimal qRT-PCR system for the first time. The stability levels of optimal and worst reference genes screened for seed development, anther sterility and plant development were validated in the qRT-PCR normalization. Our results provided a reference gene application guideline for sesame gene expression characterization using qRT-PCR system.

  11. Validation of Reference Housekeeping Genes for Gene Expression Studies in Western Corn Rootworm (Diabrotica virgifera virgifera)

    PubMed Central

    Barros Rodrigues, Thaís; Khajuria, Chitvan; Wang, Haichuan; Matz, Natalie; Cunha Cardoso, Danielle; Valicente, Fernando Hercos; Zhou, Xuguo; Siegfried, Blair

    2014-01-01

    Quantitative Real-time PCR (qRT-PCR) is a powerful technique to investigate comparative gene expression. In general, normalization of results using a highly stable housekeeping gene (HKG) as an internal control is recommended and necessary. However, there are several reports suggesting that regulation of some HKGs is affected by different conditions. The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States and Europe. The expression profile of target genes related to insecticide exposure, resistance, and RNA interference has become an important experimental technique for study of western corn rootworms; however, lack of information on reliable HKGs under different conditions makes the interpretation of qRT-PCR results difficult. In this study, four distinct algorithms (Genorm, NormFinder, BestKeeper and delta-CT) and five candidate HKGs to genes of reference (β-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; β-tubulin; RPS9, ribosomal protein S9; EF1a, elongation factor-1α) were evaluated to determine the most reliable HKG under different experimental conditions including exposure to dsRNA and Bt toxins and among different tissues and developmental stages. Although all the HKGs tested exhibited relatively stable expression among the different treatments, some differences were noted. Among the five candidate reference genes evaluated, β-actin exhibited highly stable expression among different life stages. RPS9 exhibited the most similar pattern of expression among dsRNA treatments, and both experiments indicated that EF1a was the second most stable gene. EF1a was also the most stable for Bt exposure and among different tissues. These results will enable researchers to use more accurate and reliable normalization of qRT-PCR data in WCR experiments. PMID:25356627

  12. Selection and validation of reference genes for quantitative real-time PCR analysis of gene expression in Cichorium intybus

    PubMed Central

    Delporte, Marianne; Legrand, Guillaume; Hilbert, Jean-Louis; Gagneul, David

    2015-01-01

    Plant polyphenols represent a huge reservoir of bioactive compounds. Industrial chicory, an important crop from northwestern Europe, accumulates an original combination of such compounds, i.e., chlorogenic, isochlorogenic, caftaric, and chicoric acids arising from the phenylpropanoid pathway. For a complete understanding of these biochemical pathways, analyses of gene expression using quantitative real-time PCR (qRT-PCR) should be considered. Because cell cultures are a model of choice for specialized metabolism investigations, this study described for the first time the validation of reference genes for this system in chicory. Eighteen potential reference genes were obtained by mining expressed sequence tag databases of chicory for orthologs of Arabidopsis thaliana genes currently used as reference genes. Twelve genes passed the qRT-PCR standard requirements and their expression stability across different samples was tested using three distinct softwares: geNorm, NormFinder, and BestKeeper. In cell cultures grown under various conditions, TIP41 (TIP41 like protein) was shown to be the most stable gene. Further validation of the proposed reference genes was done by normalization of expression levels of a group of genes of interest. In order to assess the potentiality of the proposed list of candidate reference genes, theses genes were in parallel tested on another experimental design, i.e., chicory seedlings. In this case, the best reference gene identified was Clath (Clathrin adaptator complex subunit). The results highlight the importance of the use of properly validated reference genes to achieve relevant interpretation of qRT-PCR analyses. Here, we provide a list of reference genes suitable for future gene expression studies in chicory. PMID:26347767

  13. RNA quality assessment: a view from plant qPCR studies.

    PubMed

    Die, Jose V; Román, Belén

    2012-10-01

    Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is probably the most common molecular technique used in transcriptome analyses today. The simplicity of the technology and associated protocols that generate results without the need to understand the underlying principles has made RT-qPCR the method of choice for RNA quantification. Rather than the 'gold standard technology' often used to describe it, the performance of RT-qPCR suffers from considerable pitfalls during general workflow. The inconsistency of conventional methods for the evaluation of RNA quality and its influence on qPCR performance as well as stability of reference genes is summarized and discussed here.

  14. Validation of reference genes for RT-qPCR analysis in Herbaspirillum seropedicae.

    PubMed

    Pessoa, Daniella Duarte Villarinho; Vidal, Marcia Soares; Baldani, José Ivo; Simoes-Araujo, Jean Luiz

    2016-08-01

    The RT-qPCR technique needs a validated set of reference genes for ensuring the consistency of the results from the gene expression. Expression stabilities for 9 genes from Herbaspirillum seropedicae, strain HRC54, grown with different carbon sources were calculated using geNorm and NormFinder, and the gene rpoA showed the best stability values.

  15. Evaluation of reference genes for gene expression in red-tailed phascogale (Phascogale calura) liver, lung, small intestine and spleen

    PubMed Central

    Ong, Oselyne T.W.; Young, Lauren J.

    2016-01-01

    Background Reference genes serve an important role as an endogenous control/standard for data normalisation in gene expression studies. Although reference genes have recently been suggested for marsupials, independent analysis of reference genes on different immune tissues is yet to be tested. Therefore, an assessment of reference genes is needed for the selection of stable, expressed genes across different marsupial tissues. Methods The study was conducted on red-tailed phascogales (Phascogale calura) using five juvenile and five adult males. The stability of five reference genes (glyceraldehyde-3-phosphate dehydrogenase, GAPDH; β-actin, ACTB; 18S rRNA, 18S; 28S rRNA, 28S; and ribosomal protein L13A, RPL13A) was investigated using SYBR Green and analysed with the geNorm application available in qBasePLUS software. Results Gene stability for juvenile and adult tissue samples combined show that GAPDH was most stable in liver and lung tissue, and 18S in small intestine and spleen. While all reference genes were suitable for small intestine and spleen tissues, all reference genes except 28S were stable for lung and only 18S and 28S were stable for liver tissue. Separating the two age groups, we found that two different reference genes were considered stable in juveniles (ACTB and GAPDH) and adults (18S and 28S), and RPL13A was not stable for juvenile small intestine tissue. Except for 28S, all reference genes were stable in juvenile and adult lungs, and all five reference genes were stable in spleen tissue. Discussion Based on expression stability, ACTB and GAPDH are suitable for all tissues when studying the expression of marsupials in two age groups, except for adult liver tissues. The expression stability between juvenile and adult liver tissue was most unstable, as the stable reference genes for juveniles and adults were different. Juvenile and adult lung, small intestine and spleen share similar stable reference genes, except for small intestine tissues where

  16. Identification and validation of reference genes for transcript normalization in strawberry (Fragaria × ananassa) defense responses.

    PubMed

    Amil-Ruiz, Francisco; Garrido-Gala, José; Blanco-Portales, Rosario; Folta, Kevin M; Muñoz-Blanco, Juan; Caballero, José L

    2013-01-01

    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.

  17. Identification and Validation of Reference Genes for Transcript Normalization in Strawberry (Fragaria × ananassa) Defense Responses

    PubMed Central

    Amil-Ruiz, Francisco; Garrido-Gala, José; Blanco-Portales, Rosario; Folta, Kevin M.; Muñoz-Blanco, Juan; Caballero, José L.

    2013-01-01

    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. PMID:23940602

  18. Bioinformatics approach for choosing the correct reference genes when studying gene expression in human keratinocytes.

    PubMed

    Beer, Lucian; Mlitz, Veronika; Gschwandtner, Maria; Berger, Tanja; Narzt, Marie-Sophie; Gruber, Florian; Brunner, Patrick M; Tschachler, Erwin; Mildner, Michael

    2015-10-01

    Reverse transcription polymerase chain reaction (qRT-PCR) has become a mainstay in many areas of skin research. To enable quantitative analysis, it is necessary to analyse expression of reference genes (RGs) for normalization of target gene expression. The selection of reliable RGs therefore has an important impact on the experimental outcome. In this study, we aimed to identify and validate the best suited RGs for qRT-PCR in human primary keratinocytes (KCs) over a broad range of experimental conditions using the novel bioinformatics tool 'RefGenes', which is based on a manually curated database of published microarray data. Expression of 6 RGs identified by RefGenes software and 12 commonly used RGs were validated by qRT-PCR. We assessed whether these 18 markers fulfilled the requirements for a valid RG by the comprehensive ranking of four bioinformatics tools and the coefficient of variation (CV). In an overall ranking, we found GUSB to be the most stably expressed RG, whereas the expression values of the commonly used RGs, GAPDH and B2M were significantly affected by varying experimental conditions. Our results identify RefGenes as a powerful tool for the identification of valid RGs and suggest GUSB as the most reliable RG for KCs.

  19. Reference gene selection for gene expression analysis of oocytes collected from dairy cattle and buffaloes during winter and summer.

    PubMed

    Macabelli, Carolina Habermann; Ferreira, Roberta Machado; Gimenes, Lindsay Unno; de Carvalho, Nelcio Antonio Tonizza; Soares, Júlia Gleyci; Ayres, Henderson; Ferraz, Márcio Leão; Watanabe, Yeda Fumie; Watanabe, Osnir Yoshime; Sangalli, Juliano Rodrigues; Smith, Lawrence Charles; Baruselli, Pietro Sampaio; Meirelles, Flávio Vieira; Chiaratti, Marcos Roberto

    2014-01-01

    Oocytes from dairy cattle and buffaloes have severely compromised developmental competence during summer. While analysis of gene expression is a powerful technique for understanding the factors affecting developmental hindrance in oocytes, analysis by real-time reverse transcription PCR (RT-PCR) relies on the correct normalization by reference genes showing stable expression. Furthermore, several studies have found that genes commonly used as reference standards do not behave as expected depending on cell type and experimental design. Hence, it is recommended to evaluate expression stability of candidate reference genes for a specific experimental condition before employing them as internal controls. In acknowledgment of the importance of seasonal effects on oocyte gene expression, the aim of this study was to evaluate the stability of expression levels of ten well-known reference genes (ACTB, GAPDH, GUSB, HIST1H2AG, HPRT1, PPIA, RPL15, SDHA, TBP and YWHAZ) using oocytes collected from different categories of dairy cattle and buffaloes during winter and summer. A normalization factor was provided for cattle (RPL15, PPIA and GUSB) and buffaloes (YWHAZ, GUSB and GAPDH) based on the expression of the three most stable reference genes in each species. Normalization of non-reference target genes by these reference genes was shown to be considerably different from normalization by less stable reference genes, further highlighting the need for careful selection of internal controls. Therefore, due to the high variability of reference genes among experimental groups, we conclude that data normalized by internal controls can be misleading and should be compared to not normalized data or to data normalized by an external control in order to better interpret the biological relevance of gene expression analysis.

  20. Reference Gene Selection for Gene Expression Analysis of Oocytes Collected from Dairy Cattle and Buffaloes during Winter and Summer

    PubMed Central

    Gimenes, Lindsay Unno; de Carvalho, Nelcio Antonio Tonizza; Soares, Júlia Gleyci; Ayres, Henderson; Ferraz, Márcio Leão; Watanabe, Yeda Fumie; Watanabe, Osnir Yoshime; Sangalli, Juliano Rodrigues; Smith, Lawrence Charles; Baruselli, Pietro Sampaio; Meirelles, Flávio Vieira; Chiaratti, Marcos Roberto

    2014-01-01

    Oocytes from dairy cattle and buffaloes have severely compromised developmental competence during summer. While analysis of gene expression is a powerful technique for understanding the factors affecting developmental hindrance in oocytes, analysis by real-time reverse transcription PCR (RT-PCR) relies on the correct normalization by reference genes showing stable expression. Furthermore, several studies have found that genes commonly used as reference standards do not behave as expected depending on cell type and experimental design. Hence, it is recommended to evaluate expression stability of candidate reference genes for a specific experimental condition before employing them as internal controls. In acknowledgment of the importance of seasonal effects on oocyte gene expression, the aim of this study was to evaluate the stability of expression levels of ten well-known reference genes (ACTB, GAPDH, GUSB, HIST1H2AG, HPRT1, PPIA, RPL15, SDHA, TBP and YWHAZ) using oocytes collected from different categories of dairy cattle and buffaloes during winter and summer. A normalization factor was provided for cattle (RPL15, PPIA and GUSB) and buffaloes (YWHAZ, GUSB and GAPDH) based on the expression of the three most stable reference genes in each species. Normalization of non-reference target genes by these reference genes was shown to be considerably different from normalization by less stable reference genes, further highlighting the need for careful selection of internal controls. Therefore, due to the high variability of reference genes among experimental groups, we conclude that data normalized by internal controls can be misleading and should be compared to not normalized data or to data normalized by an external control in order to better interpret the biological relevance of gene expression analysis. PMID:24676354

  1. Selection of Reference Genes for Gene Expression Normalization in Peucedanum praeruptorum Dunn under Abiotic Stresses, Hormone Treatments and Different Tissues.

    PubMed

    Zhao, Yucheng; Luo, Jun; Xu, Sheng; Wang, Wei; Liu, Tingting; Han, Chao; Chen, Yijun; Kong, Lingyi

    2016-01-01

    Peucedanum praeruptorum Dunn is one of the main traditional Chinese medicines producing coumarins and plenty of literatures are focused on the biosynthesis of coumarins. Quantitative real-time reverse transcription PCR (qRT-PCR) is a widely used method in studying the biosynthesis pathway and the selection of reference genes plays a crucial role in accurate normalization. To facilitate biosynthesis study of coumarins, twelve candidate reference genes were selected from the transcriptome database of P. praeruptorum according to previous studies. Then, BestKeeper, geNoFrm and NormFinder were used for selecting stably expressed reference genes in different tissues and under various stress treatments. The results indicated that, among the twelve candidate reference genes, the SAND family protein (SAND), actin 2 (ACT2), ubiquitin-conjugating enzyme 9 (UBC9), protein phosphatase 2A gene (PP2A) and polypyrimidine tract-binding protein (PTBP1) were the most stable reference genes under different experimental treatments, while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tubulin beta-6 (TUB6) were the least stable genes. In addition, the suitability of SAND, TIP41-like protein (TIP41), UBC9, ACT2, TUB6 and their combination as reference genes were confirmed by normalizing the expression of 1-aminocyclopropane-1-carboxylate oxidase (ACO) in different treatments. This work is the first survey of the stability of reference genes in P. praeruptorum and provides guidelines to obtain more accurate qRT-PCR results in P. praeruptorum and other plant species.

  2. Selection of Reference Genes for Gene Expression Normalization in Peucedanum praeruptorum Dunn under Abiotic Stresses, Hormone Treatments and Different Tissues

    PubMed Central

    Zhao, Yucheng; Luo, Jun; Xu, Sheng; Wang, Wei; Liu, Tingting; Han, Chao; Chen, Yijun; Kong, Lingyi

    2016-01-01

    Peucedanum praeruptorum Dunn is one of the main traditional Chinese medicines producing coumarins and plenty of literatures are focused on the biosynthesis of coumarins. Quantitative real-time reverse transcription PCR (qRT-PCR) is a widely used method in studying the biosynthesis pathway and the selection of reference genes plays a crucial role in accurate normalization. To facilitate biosynthesis study of coumarins, twelve candidate reference genes were selected from the transcriptome database of P. praeruptorum according to previous studies. Then, BestKeeper, geNoFrm and NormFinder were used for selecting stably expressed reference genes in different tissues and under various stress treatments. The results indicated that, among the twelve candidate reference genes, the SAND family protein (SAND), actin 2 (ACT2), ubiquitin-conjugating enzyme 9 (UBC9), protein phosphatase 2A gene (PP2A) and polypyrimidine tract-binding protein (PTBP1) were the most stable reference genes under different experimental treatments, while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tubulin beta-6 (TUB6) were the least stable genes. In addition, the suitability of SAND, TIP41-like protein (TIP41), UBC9, ACT2, TUB6 and their combination as reference genes were confirmed by normalizing the expression of 1-aminocyclopropane-1-carboxylate oxidase (ACO) in different treatments. This work is the first survey of the stability of reference genes in P. praeruptorum and provides guidelines to obtain more accurate qRT-PCR results in P. praeruptorum and other plant species. PMID:27022972

  3. Evaluation of Clostridium ljungdahlii DSM 13528 reference genes in gene expression studies by qRT-PCR.

    PubMed

    Liu, Juanjuan; Tan, Yang; Yang, Xiaohong; Chen, Xiaohua; Li, Fuli

    2013-10-01

    Clostridium ljungdahlii DSM 13528 is a promising platform organism for biofuel production from syngas. Gene expression analysis permits a better understanding of the important molecular biological characteristics of this organism, such as carbon fixation and solvent adaptation. Normalization is a prerequisite for accurate gene expression analysis, but until now, no valid reference genes have been proposed for quantitative real-time polymerase chain reaction (qRT-PCR) analysis of C. ljungdahlii DSM 13528. In this study, seven candidate reference genes (gyrA, rho, fotl, rpoA, gukl, recA, 16S rRNA) were selected for qRT-PCR quantification of their expression levels in various culture conditions that corresponded to different carbon sources and stresses. Two analytical programs, geNorm and NormFinder, were used to evaluate reference gene stability. The results showed that gyrA, rho and fotl exhibited the most stable expression levels across all tested samples and can be confidently used as reference genes to normalize the transcriptional data of target genes in qRT-PCR analyses of C. ljungdahlii DSM 13528. This study presents the first attempt to explore the validity of candidate reference genes and provide a set of valid reference genes for normalizing C. ljungdahlii DSM 13528 target gene expression and transcriptome analysis.

  4. Selection of suitable reference genes for gene expression studies in normal human ovarian tissues, borderline ovarian tumours and ovarian cancer.

    PubMed

    Ofinran, Olumide; Bose, Ujjal; Hay, Daniel; Abdul, Summi; Tufatelli, Cristina; Khan, Raheela

    2016-12-01

    The use of reference genes is the most common method of controlling the variation in mRNA expression during quantitative polymerase chain reaction, although the use of traditional reference genes, such as β‑actin, glyceraldehyde‑3‑phosphate dehydrogenase or 18S ribosomal RNA, without validation occasionally leads to unreliable results. Therefore, the present study aimed to evaluate a set of five commonly used reference genes to determine the most suitable for gene expression studies in normal ovarian tissues, borderline ovarian and ovarian cancer tissues. The expression stabilities of these genes were ranked using two gene stability algorithms, geNorm and NormFinder. Using geNorm, the two best reference genes in ovarian cancer were β‑glucuronidase and β‑actin. Hypoxanthine phosphoribosyltransferase‑1 and β‑glucuronidase were the most stable in ovarian borderline tumours, and hypoxanthine phosphoribosyltransferase‑1 and glyceraldehyde‑3‑phosphate dehydrogenase were the most stable in normal ovarian tissues. NormFinder ranked β‑actin the most stable in ovarian cancer, and the best combination of two genes was β‑glucuronidase and β‑actin. In borderline tumours, hypoxanthine phosphoribosyltransferase‑1 was identified as the most stable, and the best combination was hypoxanthine phosphoribosyltransferase‑1 and β‑glucuronidase. In normal ovarian tissues, β‑glucuronidase was recommended as the optimum reference gene, and the most optimum pair of reference genes was hypoxanthine phosphoribosyltransferase‑1 and β‑actin. To the best of our knowledge, this is the first study to investigate the selection of a set of reference genes for normalisation in quantitative polymerase chain reactions in different ovarian tissues, and therefore it is recommended that β‑glucuronidase, β‑actin and hypoxanthine phosphoribosyltransferase‑1 are the most suitable reference genes for such analyses.

  5. Selection of suitable reference genes for gene expression studies in Staphylococcus capitis during growth under erythromycin stress.

    PubMed

    Cui, Bintao; Smooker, Peter M; Rouch, Duncan A; Deighton, Margaret A

    2016-08-01

    Accurate and reproducible measurement of gene transcription requires appropriate reference genes, which are stably expressed under different experimental conditions to provide normalization. Staphylococcus capitis is a human pathogen that produces biofilm under stress, such as imposed by antimicrobial agents. In this study, a set of five commonly used staphylococcal reference genes (gyrB, sodA, recA, tuf and rpoB) were systematically evaluated in two clinical isolates of Staphylococcus capitis (S. capitis subspecies urealyticus and capitis, respectively) under erythromycin stress in mid-log and stationary phases. Two public software programs (geNorm and NormFinder) and two manual calculation methods, reference residue normalization (RRN) and relative quantitative (RQ), were applied. The potential reference genes selected by the four algorithms were further validated by comparing the expression of a well-studied biofilm gene (icaA) with phenotypic biofilm formation in S. capitis under four different experimental conditions. The four methods differed considerably in their ability to predict the most suitable reference gene or gene combination for comparing icaA expression under different conditions. Under the conditions used here, the RQ method provided better selection of reference genes than the other three algorithms; however, this finding needs to be confirmed with a larger number of isolates. This study reinforces the need to assess the stability of reference genes for analysis of target gene expression under different conditions and the use of more than one algorithm in such studies. Although this work was conducted using a specific human pathogen, it emphasizes the importance of selecting suitable reference genes for accurate normalization of gene expression more generally.

  6. Expression Profiling in Bemisia tabaci under Insecticide Treatment: Indicating the Necessity for Custom Reference Gene Selection

    PubMed Central

    Zhou, Xuguo; Gao, Xiwu

    2014-01-01

    Finding a suitable reference gene is the key for qRT-PCR analysis. However, none of the reference gene discovered thus far can be utilized universally under various biotic and abiotic experimental conditions. In this study, we further examine the stability of candidate reference genes under a single abiotic factor, insecticide treatment. After being exposed to eight commercially available insecticides, which belong to five different classes, the expression profiles of eight housekeeping genes in the sweetpotato whitefly, Bemisia tabaci, one of the most invasive and destructive pests in the world, were investigated using qRT-PCR analysis. In summary, elongation factor 1α (EF1α), α-tubulin (TUB1α) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified as the most stable reference genes under the insecticide treatment. The initial assessment of candidate reference genes was further validated with the expression of two target genes, a P450 (Cyp6cm1) and a glutathione S-transferase (GST). However, ranking of reference genes varied substantially among intra- and inter-classes of insecticides. These combined data strongly suggested the necessity of conducting custom reference gene selection designed for each and every experimental condition, even when examining the same abiotic or biotic factor. PMID:24498122

  7. Reference gene selection for qRT-PCR in Caragana korshinskii Kom. under different stress conditions.

    PubMed

    Yang, Qi; Yin, Jiajia; Li, Gao; Qi, Liwang; Yang, Feiyun; Wang, Ruigang; Li, Guojing

    2014-01-01

    Caragana korshinskii Kom., which is widely distributed in the northwest China and Mongolia, is an important forage bush belonging to the legume family with high economic and ecological value. Strong tolerance ability to various stresses makes C. korshinskii Kom. a valuable species for plant stress research. In this study, suitable reference genes for quantitative real-time reverse transcription PCR (qRT-PCR) were screened from 11 candidate reference genes, including ACT, GAPDH, EF1α, UBQ, TUA, CAP, TUB, TUB3, SKIP1, SKIP5-1 and SKIP5-2. A total of 129 samples under drought, heat, cold, salt, ABA and high pH treatment were profiled, and software such as geNORM, NormFinder and BestKeeper were used for reference gene evaluation and selection. Different suitable reference genes were selected under different stresses. Across all 129 samples, GAPDH, EF1α and SKIP5-1 were found to be the most stable reference genes, and EF1α+SKIP5-1 is the most stable reference gene combination. Conversely, TUA, TUB and SKIP1 were not suitable for using as reference genes owing to their great expression variation under some stress conditions. The relative expression levels of CkWRKY1 were detected using the stable and unstable reference genes and their applicability was confirmed. These results provide some stable reference genes and reference gene combinations for qRT-PCR under different stresses in C. korshinskii Kom. for future research work, and indicate that CkWRKY1 plays essential roles in response to stresses in C. korshinskii.

  8. Selection of low-variance expressed Malus x domestica (apple) genes for use as quantitative PCR reference genes (housekeepers)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To accurately measure gene expression using PCR-based approaches, there is the need for reference genes that have low variance in expression (housekeeping genes) to normalise the data for RNA quantity and quality. For non-model species such as Malus x domestica (apples), previously, the selection of...

  9. A unified gene catalog for the laboratory mouse reference genome.

    PubMed

    Zhu, Y; Richardson, J E; Hale, P; Baldarelli, R M; Reed, D J; Recla, J M; Sinclair, R; Reddy, T B K; Bult, C J

    2015-08-01

    We report here a semi-automated process by which mouse genome feature predictions and curated annotations (i.e., genes, pseudogenes, functional RNAs, etc.) from Ensembl, NCBI and Vertebrate Genome Annotation database (Vega) are reconciled with the genome features in the Mouse Genome Informatics (MGI) database (http://www.informatics.jax.org) into a comprehensive and non-redundant catalog. Our gene unification method employs an algorithm (fjoin--feature join) for efficient detection of genome coordinate overlaps among features represented in two annotation data sets. Following the analysis with fjoin, genome features are binned into six possible categories (1:1, 1:0, 0:1, 1:n, n:1, n:m) based on coordinate overlaps. These categories are subsequently prioritized for assessment of annotation equivalencies and differences. The version of the unified catalog reported here contains more than 59,000 entries, including 22,599 protein-coding coding genes, 12,455 pseudogenes, and 24,007 other feature types (e.g., microRNAs, lincRNAs, etc.). More than 23,000 of the entries in the MGI gene catalog have equivalent gene models in the annotation files obtained from NCBI, Vega, and Ensembl. 12,719 of the features are unique to NCBI relative to Ensembl/Vega; 11,957 are unique to Ensembl/Vega relative to NCBI, and 3095 are unique to MGI. More than 4000 genome features fall into categories that require manual inspection to resolve structural differences in the gene models from different annotation sources. Using the MGI unified gene catalog, researchers can easily generate a comprehensive report of mouse genome features from a single source and compare the details of gene and transcript structure using MGI's mouse genome browser.

  10. Identification and validation of reference genes for quantitative RT-PCR normalization in wheat

    PubMed Central

    Paolacci, Anna R; Tanzarella, Oronzo A; Porceddu, Enrico; Ciaffi, Mario

    2009-01-01

    Background Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR. Results 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. Conclusion 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

  11. Evaluation of endogenous reference genes for analysis of gene expression with real-time RT-PCR during planarian regeneration.

    PubMed

    Yuwen, Yan-Qing; Dong, Zi-Mei; Wang, Qing-Hua; Sun, Xiao-Juan; Shi, Chang-Ying; Chen, Guang-Wen

    2011-10-01

    It is important that endogenous reference genes for real-time RT-PCR be empirically evaluated for stability in different cell types, developmental stages, and/or sample treatment. To select the most stable endogenous reference genes during planarian regeneration, three housekeeping genes, 18S rRNA, ACTB and DjEF2, were identified and established expression levels by real-time RT-PCR. The data were analyzed by GeNorm and NormFinder software. Expression levels of the Djsix-1 gene were studied in parallel with ACTB and DjEF2 both or each and 18S rRNA as reference during regeneration. The results showed that ACTB was the most stable expressed reference gene in the planarian regeneration.

  12. Identification of internal reference genes for gene expression normalization between the two sexes in dioecious white Campion.

    PubMed

    Zemp, Niklaus; Minder, Aria; Widmer, Alex

    2014-01-01

    Quantitative real time (qRT)-PCR is a precise and efficient method for studying gene expression changes between two states of interest, and is frequently used for validating interesting gene expression patterns in candidate genes initially identified in genome-wide expression analyses, such as RNA-seq experiments. For an adequate normalisation of qRT-PCR data, it is essential to have reference genes available whose expression intensities are constant among the different states of interest. In this study we present and validate a catalogue of traditional and newly identified reference genes that were selected from RNA-seq data from multiple individuals from the dioecious plant Silene latifolia with the aim of studying gene expression differences between the two sexes in both reproductive and vegetative tissues. The catalogue contains more than 15 reference genes with both stable expression intensities and a range of expression intensities in flower buds and leaf tissues. These reference genes were used to normalize expression differences between reproductive and vegetative tissues in eight candidate genes with sex-biased expression. Our results suggest a trend towards a reduced sex-bias in sex-linked gene expression in vegetative tissues. In this study, we report on the systematic identification and validation of internal reference genes for adequate normalization of qRT-PCR-based analyses of gene expression differences between the two sexes in S. latifolia. We also show how RNA-seq data can be used efficiently to identify suitable reference genes in a wide diversity of species.

  13. Mrpl10 and Tbp Are Suitable Reference Genes for Peripheral Nerve Crush Injury

    PubMed Central

    Wang, Yaxian; Shan, Qianqian; Meng, Yali; Pan, Jiacheng; Yi, Sheng

    2017-01-01

    Peripheral nerve injury triggers the dysregulation of a large number of genes at multiple sites, including neurons, peripheral nerve stump, and the target organ. Housekeeping genes were frequently used as reference genes to normalize the expression values of target genes. Suitable selection of housekeeping genes that are stably expressed after nerve injury minimizes bias elicited by reference genes and thus helps to better and more sensitively reflect gene expression changes. However, many housekeeping genes have been used as reference genes without testing the expression patterns of themselves. In the current study, we calculated the expression stability of nine commonly used housekeeping genes, such as 18S (18S ribosomal RNA), Actb (β-actin), CypA (cyclophilin A), Gapdh (glyceraldehydes-3-phosphate dehydrogenase), Hprt (hypoxanthine guanine phosphoribosyl transferase), Pgk1 (phosphoglycerate kinase 1), Tbp (TATA box binding protein), Ubc (ubiquitin C), YwhaZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation), and four newly identified housekeeping genes, including Ankrd27 (Ankyrin repeat domain 27), Mrpl10 (mitochondrial ribosomal protein L10), Rictor (rapamycin-insensitive companion of mTOR, Complex 2), and Ubxn 11 (UBX domain protein 11), in both distal sciatic nerve samples and dorsal root ganglion (DRG) samples after sciatic nerve injury. Our results suggested that following peripheral nerve injury, Mrpl10 and Tbp might be used as suitable reference genes for sciatic nerve stump and DRGs, respectively. PMID:28134789

  14. A reference gene set for chemosensory receptor genes of Manduca sexta.

    PubMed

    Koenig, Christopher; Hirsh, Ariana; Bucks, Sascha; Klinner, Christian; Vogel, Heiko; Shukla, Aditi; Mansfield, Jennifer H; Morton, Brian; Hansson, Bill S; Grosse-Wilde, Ewald

    2015-11-01

    The order of Lepidoptera has historically been crucial for chemosensory research, with many important advances coming from the analysis of species like Bombyx mori or the tobacco hornworm, Manduca sexta. Specifically M. sexta has long been a major model species in the field, especially regarding the importance of olfaction in an ecological context, mainly the interaction with its host plants. In recent years transcriptomic data has led to the discovery of members of all major chemosensory receptor families in the species, but the data was fragmentary and incomplete. Here we present the analysis of the newly available high-quality genome data for the species, supplemented by additional transcriptome data to generate a high quality reference gene set for the three major chemosensory receptor gene families, the gustatory (GR), olfactory (OR) and antennal ionotropic receptors (IR). Coupled with gene expression analysis our approach allows association of specific receptor types and behaviors, like pheromone and host detection. The dataset will provide valuable support for future analysis of these essential chemosensory modalities in this species and in Lepidoptera in general.

  15. Selection of suitable reference genes for assessing gene expression in pearl millet under different abiotic stresses and their combinations.

    PubMed

    Shivhare, Radha; Lata, Charu

    2016-03-14

    Pearl millet [Pennisetum glaucum (L.) R. Br.] a widely used grain and forage crop, is grown in areas frequented with one or more abiotic stresses, has superior drought and heat tolerance and considered a model crop for stress tolerance studies. Selection of suitable reference genes for quantification of target stress-responsive gene expression through quantitative real-time (qRT)-PCR is important for elucidating the molecular mechanisms of improved stress tolerance. For precise normalization of gene expression data in pearl millet, ten candidate reference genes were examined in various developmental tissues as well as under different individual abiotic stresses and their combinations at 1 h (early) and 24 h (late) of stress using geNorm, NormFinder and RefFinder algorithms. Our results revealed EF-1α and UBC-E2 as the best reference genes across all samples, the specificity of which was confirmed by assessing the relative expression of a PgAP2 like-ERF gene that suggested use of these two reference genes is sufficient for accurate transcript normalization under different stress conditions. To our knowledge this is the first report on validation of reference genes under different individual and multiple abiotic stresses in pearl millet. The study can further facilitate fastidious discovery of stress-tolerance genes in this important stress-tolerant crop.

  16. Selection of suitable reference genes for assessing gene expression in pearl millet under different abiotic stresses and their combinations

    PubMed Central

    Shivhare, Radha; Lata, Charu

    2016-01-01

    Pearl millet [Pennisetum glaucum (L.) R. Br.] a widely used grain and forage crop, is grown in areas frequented with one or more abiotic stresses, has superior drought and heat tolerance and considered a model crop for stress tolerance studies. Selection of suitable reference genes for quantification of target stress-responsive gene expression through quantitative real-time (qRT)-PCR is important for elucidating the molecular mechanisms of improved stress tolerance. For precise normalization of gene expression data in pearl millet, ten candidate reference genes were examined in various developmental tissues as well as under different individual abiotic stresses and their combinations at 1 h (early) and 24 h (late) of stress using geNorm, NormFinder and RefFinder algorithms. Our results revealed EF-1α and UBC-E2 as the best reference genes across all samples, the specificity of which was confirmed by assessing the relative expression of a PgAP2 like-ERF gene that suggested use of these two reference genes is sufficient for accurate transcript normalization under different stress conditions. To our knowledge this is the first report on validation of reference genes under different individual and multiple abiotic stresses in pearl millet. The study can further facilitate fastidious discovery of stress-tolerance genes in this important stress-tolerant crop. PMID:26972345

  17. Characterization of reference gene expression in tung tree (Vernicia fordii)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tung oil from tung tree (Vernicia fordii) is widely used as a drying ingredient in paints, varnishes, and other coatings and finishes. Recent research has focused on the understanding of the biosynthesis of oil in tung trees. Many oil biosynthetic genes have been identified in tung tree but little...

  18. Identification and Characterization of Reference Genes for Normalizing Expression Data from Red Swamp Crawfish Procambarus clarkii.

    PubMed

    Jiang, Hucheng; Qian, Zhaojun; Lu, Wei; Ding, Huaiyu; Yu, Hongwei; Wang, Hui; Li, Jiale

    2015-09-08

    qRT-PCR is a widely used technique for rapid and accurate quantification of gene expression data. The use of reference genes for normalization of the expression levels is crucial for accuracy. Several studies have shown that there is no perfect reference gene that is appropriate for use in all experimental conditions, and research on suitable reference genes in red swamp crawfish (Procambarus clarkii) is particularly scarce. In this study, eight commonly used crustacean reference genes were chosen from P. clarkii transcriptome data and investigated as potential candidates for normalization of qRT-PCR data. Expression of these genes under different experimental conditions was examined by qRT-PCR, and the stability of their expression was evaluated using three commonly used statistical algorithms, geNorm, NormFinder and BestKeeper. A final comprehensive ranking determined that EIF and 18S were the optimal reference genes for expression data from different tissues, while TBP and EIF were optimal for expression data from different ovarian developmental stages. To our knowledge, this is the first systematic analysis of reference genes for normalization of qRT-PCR data in P. clarkii. These results will facilitate more accurate and reliable expression studies of this and other crustacean species.

  19. Identification of Phosphoglycerate Kinase 1 (PGK1) as a reference gene for quantitative gene expression measurements in human blood RNA

    PubMed Central

    2011-01-01

    Background Blood is a convenient sample and increasingly used for quantitative gene expression measurements with a variety of diseases including chronic fatigue syndrome (CFS). Quantitative gene expression measurements require normalization of target genes to reference genes that are stable and independent from variables being tested in the experiment. Because there are no genes that are useful for all situations, reference gene selection is an essential step to any quantitative reverse transcription-PCR protocol. Many publications have described appropriate genes for a wide variety of tissues and experimental conditions, however, reference genes that may be suitable for the analysis of CFS, or human blood RNA derived from whole blood as well as isolated peripheral blood mononuclear cells (PBMCs), have not been described. Findings Literature review and analyses of our unpublished microarray data were used to narrow down the pool of candidate reference genes to six. We assayed whole blood RNA from Tempus tubes and cell preparation tube (CPT)-collected PBMC RNA from 46 subjects, and used the geNorm and NormFinder algorithms to select the most stable reference genes. Phosphoglycerate kinase 1 (PGK1) was one of the optimal normalization genes for both whole blood and PBMC RNA, however, additional genes differed for the two sample types; Ribosomal protein large, P0 (RPLP0) for PBMC RNA and Peptidylprolyl isomerase B (PPIB) for whole blood RNA. We also show that the use of a single reference gene is sufficient for normalization when the most stable candidates are used. Conclusions We have identified PGK1 as a stable reference gene for use with whole blood RNA and RNA derived from PBMC. When stable genes are selected it is possible to use a single gene for normalization rather than two or three. Optimal normalization will improve the ability of results from PBMC RNA to be compared with those from whole blood RNA and potentially allows comparison of gene expression results

  20. Assessment of reference genes for reliable analysis of gene transcription by RT-qPCR in ovine leukocytes.

    PubMed

    Mahakapuge, T A N; Scheerlinck, J-P Y; Rojas, C A Alvarez; Every, A L; Hagen, J

    2016-03-01

    With the availability of genetic sequencing data, quantitative reverse transcription PCR (RT-qPCR) is increasingly being used for the quantification of gene transcription across species. Too often there is little regard to the selection of reference genes and the impact that a poor choice has on data interpretation. Indeed, RT-qPCR provides a snapshot of relative gene transcription at a given time-point, and hence is highly dependent on the stability of the transcription of the reference gene(s). Using ovine efferent lymph cells and peripheral blood mono-nuclear cells (PBMCs), the two most frequently used leukocytes in immunological studies, we have compared the stability of transcription of the most commonly used ovine reference genes: YWHAZ, RPL-13A, PGK1, B2M, GAPDH, HPRT, SDHA and ACTB. Using established algorithms for reference gene normalization "geNorm" and "Norm Finder", PGK1, GAPDH and YWHAZ were deemed the most stably transcribed genes for efferent leukocytes and PGK1, YWHAZ and SDHA were optimal in PBMCs. These genes should therefore be considered for accurate and reproducible RT-qPCR data analysis of gene transcription in sheep.

  1. Selection and validation of a set of reliable reference genes for quantitative RT-PCR studies in the brain of the Cephalopod Mollusc Octopus vulgaris

    PubMed Central

    Sirakov, Maria; Zarrella, Ilaria; Borra, Marco; Rizzo, Francesca; Biffali, Elio; Arnone, Maria Ina; Fiorito, Graziano

    2009-01-01

    Background Quantitative real-time polymerase chain reaction (RT-qPCR) is valuable for studying the molecular events underlying physiological and behavioral phenomena. Normalization of real-time PCR data is critical for a reliable mRNA quantification. Here we identify reference genes to be utilized in RT-qPCR experiments to normalize and monitor the expression of target genes in the brain of the cephalopod mollusc Octopus vulgaris, an invertebrate. Such an approach is novel for this taxon and of advantage in future experiments given the complexity of the behavioral repertoire of this species when compared with its relatively simple neural organization. Results We chose 16S, and 18S rRNA, actB, EEF1A, tubA and ubi as candidate reference genes (housekeeping genes, HKG). The expression of 16S and 18S was highly variable and did not meet the requirements of candidate HKG. The expression of the other genes was almost stable and uniform among samples. We analyzed the expression of HKG into two different set of animals using tissues taken from the central nervous system (brain parts) and mantle (here considered as control tissue) by BestKeeper, geNorm and NormFinder. We found that HKG expressions differed considerably with respect to brain area and octopus samples in an HKG-specific manner. However, when the mantle is treated as control tissue and the entire central nervous system is considered, NormFinder revealed tubA and ubi as the most suitable HKG pair. These two genes were utilized to evaluate the relative expression of the genes FoxP, creb, dat and TH in O. vulgaris. Conclusion We analyzed the expression profiles of some genes here identified for O. vulgaris by applying RT-qPCR analysis for the first time in cephalopods. We validated candidate reference genes and found the expression of ubi and tubA to be the most appropriate to evaluate the expression of target genes in the brain of different octopuses. Our results also underline the importance of choosing a proper

  2. Identification and evaluation of reference genes for qRT-PCR normalization in Ganoderma lucidum.

    PubMed

    Xu, Jiang; Xu, ZhiChao; Zhu, YingJie; Luo, HongMei; Qian, Jun; Ji, AiJia; Hu, YuanLei; Sun, Wei; Wang, Bo; Song, JingYuan; Sun, Chao; Chen, ShiLin

    2014-01-01

    Quantitative real-time reverse transcription PCR (qRT-PCR) is a rapid, sensitive, and reliable technique for gene expression studies. The accuracy and reliability of qRT-PCR results depend on the stability of the reference genes used for gene normalization. Therefore, a systematic process of reference gene evaluation is needed. Ganoderma lucidum is a famous medicinal mushroom in East Asia. In the current study, 10 potential reference genes were selected from the G. lucidum genomic data. The sequences of these genes were manually curated, and primers were designed following strict criteria. The experiment was conducted using qRT-PCR, and the stability of each candidate gene was assessed using four commonly used statistical programs-geNorm, NormFinder, BestKeeper, and RefFinder. According to our results, PP2A was expressed at the most stable levels under different fermentation conditions, and RPL4 was the most stably expressed gene in different tissues. RPL4, PP2A, and β-tubulin are the most commonly recommended reference genes for normalizing gene expression in the entire sample set. The current study provides a foundation for the further use of qRT-PCR in G. lucidum gene analysis.

  3. Selection of Reference Genes for Expression Studies of Xenobiotic Adaptation in Tetranychus urticae

    PubMed Central

    Morales, Mariany Ashanty; Mendoza, Bianca Marie; Lavine, Laura Corley; Lavine, Mark Daniel; Walsh, Douglas Bruce; Zhu, Fang

    2016-01-01

    Quantitative real-time PCR (qRT-PCR) is an extensively used, high-throughput method to analyze transcriptional expression of genes of interest. An appropriate normalization strategy with reliable reference genes is required for calculating gene expression across diverse experimental conditions. In this study, we aim to identify the most stable reference genes for expression studies of xenobiotic adaptation in Tetranychus urticae, an extremely polyphagous herbivore causing significant yield reduction of agriculture. We chose eight commonly used housekeeping genes as candidates. The qRT-PCR expression data for these genes were evaluated from seven populations: a susceptible and three acaricide resistant populations feeding on lima beans, and three other susceptible populations which had been shifted host from lima beans to three other plant species. The stability of the candidate reference genes was then assessed using four different algorithms (comparative ΔCt method, geNorm, NormFinder, and BestKeeper). Additionally, we used an online web-based tool (RefFinder) to assign an overall final rank for each candidate gene. Our study found that CycA and Rp49 are best for investigating gene expression in acaricide susceptible and resistant populations. GAPDH, Rp49, and Rpl18 are best for host plant shift studies. And GAPDH and Rp49 were the most stable reference genes when investigating gene expression under changes in both experimental conditions. These results will facilitate research in revealing molecular mechanisms underlying the xenobiotic adaptation of this notorious agricultural pest. PMID:27570487

  4. Selection of Candidate Reference Genes for Gene Expression Analysis in Kentucky Bluegrass (Poa pratensis L.) under Abiotic Stress.

    PubMed

    Niu, Kuiju; Shi, Yi; Ma, Huiling

    2017-01-01

    Kentucky bluegrass (Poa pratensis L.) belong to Gramineae and is widely used in lawns, golf courses, landscapes, and sport fields as a prominent cool-season grass. Gene expression patterns during different stages of plant development can provide clues toward the understanding of its biological functions. The selection and validation of reference genes are the first steps in any real-time quantitative PCR gene expression study. Therefore, suitable reference genes are necessary for obtaining reliable results in real-time quantitative PCR analyses of Kentucky bluegrass. In the present study, 9 candidate reference genes were chosen, and their expression stability in the leaves and roots of Kentucky bluegrass under different stresses (drought, salt, heat, and cold) were evaluated using the GeNorm, NormFinder, BestKeeper, and RefFinder programs. The results showed that the expression stability of the candidate reference genes was dependent on the experimental conditions. The combination of SAM with GAPDH was the most stable in leaves under salt stress and cold stress, while TUB combined with ACT or GAPDH was stable in roots under salt or cold stress, respectively. ACT and SAM maintained stable expression in drought-treated leaves, and GAPDH combined with ACT was stable in drought-treated roots. SAM and TUB exhibited stable expression in heat-treated leaves. ACT and RPL were stable in heat-treated roots. In addition, the expression patterns of PpFEH in response to drought and cold stress were used to confirm the reliability of the selected reference genes, indicating that the use of an inappropriate reference gene as the internal control will cause erroneous results. This work is the first study on the expression stability of reference genes in Kentucky bluegrass and will be particularly useful in the selection of stress-tolerance genes and the identification of the molecular mechanisms conferring stress tolerance in this species.

  5. Selection of Candidate Reference Genes for Gene Expression Analysis in Kentucky Bluegrass (Poa pratensis L.) under Abiotic Stress

    PubMed Central

    Niu, Kuiju; Shi, Yi; Ma, Huiling

    2017-01-01

    Kentucky bluegrass (Poa pratensis L.) belong to Gramineae and is widely used in lawns, golf courses, landscapes, and sport fields as a prominent cool-season grass. Gene expression patterns during different stages of plant development can provide clues toward the understanding of its biological functions. The selection and validation of reference genes are the first steps in any real-time quantitative PCR gene expression study. Therefore, suitable reference genes are necessary for obtaining reliable results in real-time quantitative PCR analyses of Kentucky bluegrass. In the present study, 9 candidate reference genes were chosen, and their expression stability in the leaves and roots of Kentucky bluegrass under different stresses (drought, salt, heat, and cold) were evaluated using the GeNorm, NormFinder, BestKeeper, and RefFinder programs. The results showed that the expression stability of the candidate reference genes was dependent on the experimental conditions. The combination of SAM with GAPDH was the most stable in leaves under salt stress and cold stress, while TUB combined with ACT or GAPDH was stable in roots under salt or cold stress, respectively. ACT and SAM maintained stable expression in drought-treated leaves, and GAPDH combined with ACT was stable in drought-treated roots. SAM and TUB exhibited stable expression in heat-treated leaves. ACT and RPL were stable in heat-treated roots. In addition, the expression patterns of PpFEH in response to drought and cold stress were used to confirm the reliability of the selected reference genes, indicating that the use of an inappropriate reference gene as the internal control will cause erroneous results. This work is the first study on the expression stability of reference genes in Kentucky bluegrass and will be particularly useful in the selection of stress-tolerance genes and the identification of the molecular mechanisms conferring stress tolerance in this species. PMID:28261247

  6. Identification of a novel reference gene for apple transcriptional profiling under postharvest conditions.

    PubMed

    Storch, Tatiane Timm; Pegoraro, Camila; Finatto, Taciane; Quecini, Vera; Rombaldi, Cesar Valmor; Girardi, César Luis

    2015-01-01

    Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression profiles of the genes of interest against those of constitutively expressed, reference genes. Although the technique is widely used in fruit postharvest experiments, the transcription stability of reference genes has not been thoroughly investigated under these experimental conditions. Thus, we have determined the transcriptional profile, under these conditions, of three genes commonly used as reference--ACTIN (MdACT), PROTEIN DISULPHIDE ISOMERASE (MdPDI) and UBIQUITIN-CONJUGATING ENZYME E2 (MdUBC)--along with two novel candidates--HISTONE 1 (MdH1) and NUCLEOSSOME ASSEMBLY 1 PROTEIN (MdNAP1). The expression profile of the genes was investigated throughout five experiments, with three of them encompassing the postharvest period and the other two, consisting of developmental and spatial phases. The transcriptional stability was comparatively investigated using four distinct software packages: BestKeeper, NormFinder, geNorm and DataAssist. Gene ranking results for transcriptional stability were similar for the investigated software packages, with the exception of BestKeeper. The classic reference gene MdUBC ranked among the most stably transcribed in all investigated experimental conditions. Transcript accumulation profiles for the novel reference candidate gene MdH1 were stable throughout the tested conditions, especially in experiments encompassing the postharvest period. Thus, our results present a novel reference gene for postharvest experiments in apple and reinforce the importance of checking the transcription profile of reference genes under the experimental conditions of interest.

  7. Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis

    PubMed Central

    Yan, Zhaoping; Gao, Jinhang; Lv, Xiuhe; Yang, Wenjuan; Wen, Shilei; Tong, Huan; Tang, Chengwei

    2016-01-01

    The analysis of differences in gene expression is dependent on normalization using reference genes. However, the expression of many of these reference genes, as evaluated by quantitative RT-PCR, is upregulated in acute pancreatitis, so they cannot be used as the standard for gene expression in this condition. For this reason, we sought to identify a stable reference gene, or a suitable combination, for expression analysis in acute pancreatitis. The expression stability of 10 reference genes (ACTB, GAPDH, 18sRNA, TUBB, B2M, HPRT1, UBC, YWHAZ, EF-1α, and RPL-13A) was analyzed using geNorm, NormFinder, and BestKeeper software and evaluated according to variations in the raw Ct values. These reference genes were evaluated using a comprehensive method, which ranked the expression stability of these genes as follows (from most stable to least stable): RPL-13A, YWHAZ > HPRT1 > GAPDH > UBC > EF-1α > 18sRNA > B2M > TUBB > ACTB. RPL-13A was the most suitable reference gene, and the combination of RPL-13A and YWHAZ was the most stable group of reference genes in our experiments. The expression levels of ACTB, TUBB, and B2M were found to be significantly upregulated during acute pancreatitis, whereas the expression level of 18sRNA was downregulated. Thus, we recommend the use of RPL-13A or a combination of RPL-13A and YWHAZ for normalization in qRT-PCR analyses of gene expression in mouse models of acute pancreatitis. PMID:27069927

  8. Screening for Suitable Reference Genes for Quantitative Real-Time PCR in Heterosigma akashiwo (Raphidophyceae).

    PubMed

    Ji, Nanjing; Li, Ling; Lin, Lingxiao; Lin, Senjie

    2015-01-01

    The raphidophyte Heterosigma akashiwo is a globally distributed harmful alga that has been associated with fish kills in coastal waters. To understand the mechanisms of H. akashiwo bloom formation, gene expression analysis is often required. To accurately characterize the expression levels of a gene of interest, proper reference genes are essential. In this study, we assessed ten of the previously reported algal candidate genes (rpL17-2, rpL23, cox2, cal, tua, tub, ef1, 18S, gapdh, and mdh) for their suitability as reference genes in this species. We used qRT-PCR to quantify the expression levels of these genes in H. akashiwo grown under different temperatures, light intensities, nutrient concentrations, and time points over a diel cycle. The expression stability of these genes was evaluated using geNorm and NormFinder algorithms. Although none of these genes exhibited invariable expression levels, cal, tub, rpL17-2 and rpL23 expression levels were the most stable across the different conditions tested. For further validation, these selected genes were used to normalize the expression levels of ribulose-1, 5-bisphosphate carboxylase/oxygenase large unite (HrbcL) over a diel cycle. Results showed that the expression of HrbcL normalized against each of these reference genes was the highest at midday and lowest at midnight, similar to the diel patterns typically documented for this gene in algae. While the validated reference genes will be useful for future gene expression studies on H. akashiwo, we expect that the procedure used in this study may be helpful to future efforts to screen reference genes for other algae.

  9. Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR.

    PubMed

    Zampieri, Denise; Nora, Luísa C; Basso, Vanessa; Camassola, Marli; Dillon, Aldo J P

    2014-08-01

    Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a methodology that facilitates the quantification of mRNA expression in a given sample. Analysis of relative gene expression by qRT-PCR requires normalization of the data using a reference gene that is expressed at a similar level in all evaluated conditions. Determining an internal control gene is essential for gene expression studies. Gene expression studies in filamentous fungi frequently use the β-actin gene (actb), β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase as reference genes because they are known to have consistent expression levels. Until now, no study has been performed to select an internal control gene for the filamentous fungal species Penicillium echinulatum. The aim of this study was to evaluate and validate internal control genes to enable the study of gene expression in P. echinulatum using qRT-PCR. P. echinulatum strain S1M29 was grown in conditions to either induce (cellulose and sugar cane bagasse) or repress (glucose) gene expression to analyze 23 candidate normalization genes for stable expression. Two software programs, BestKeeper and geNorm, were used to assess the expression of the candidate normalization genes. The results indicate that the actb reference gene is more stably expressed in P. echinulatum. This is the first report in the literature that determines a normalization gene for this fungus. From the results obtained, we recommend the use of the P. echinulatum actb gene as an endogenous control for gene expression studies of cellulases and hemicellulases by qRT-PCR.

  10. Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

    NASA Astrophysics Data System (ADS)

    Zhao, Ye; Chen, Muyan; Wang, Tianming; Sun, Lina; Xu, Dongxue; Yang, Hongsheng

    2014-11-01

    Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues (intestine, respiratory tree, and muscle) of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the β-tubulin (TUBB) gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 (RPS18), TUBB, and NADH dehydrogenase (NADH); for respiratory tree, it was β-actin (ACTB), TUBB, and succinate dehydrogenase cytochrome B small subunit (SDHC); and for muscle it was α-tubulin (TUBA) and NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 13 (NDUFA13). These combinations of internal control genes should be considered for use in further studies of gene expression in A. japonicus during aestivation.

  11. Selection of Reference Genes for Real-Time Quantitative PCR in Pinus massoniana Post Nematode Inoculation

    PubMed Central

    Wei, Yongcheng; Liu, Qinghua; Dong, Hongyu; Zhou, Zhichun; Hao, Yanping; Chen, Xuelian; Xu, Liuyi

    2016-01-01

    Pinus massoniaia Lamb has gained more and more attention as the most important tree species for timber and forestation in South China. Gene expression studies are of great importance to identify new and elite cultivars. Real-time quantitative PCR, a highly sensitive and specific method, is commonly used in the analysis of gene expression. The appropriate reference genes must be employed to normalize the calculation program for ascertaining repeatable and significant results. Herein, eleven housekeeping genes were evaluated during different stages of P. massoniana post nematode inoculation in this study. Three statistical approaches such as geNorm, NormFinder and BestKeeper were selected to analyze the stability of candidate genes. The results indicated that U2af and β-TUB were the most stable reference genes. These two genes could be used for the normalization in most of the experiments of P. massoniana, while Histone and AK were the least stable ones. In addition, EF expressed at the lowest average Ct value was the most abundant candidate gene. As an important gene associated with defense mechanisms, ABC transporter was analyzed by qRT-PCR, and the results were used to confirm the reliability of two genes. The selected reference genes in the present study will be conducive to future gene expression normalized by qRT-PCR in P. massoniana. PMID:26800152

  12. Selection of Reference Genes for Real-Time Quantitative PCR in Pinus massoniana Post Nematode Inoculation.

    PubMed

    Wei, Yongcheng; Liu, Qinghua; Dong, Hongyu; Zhou, Zhichun; Hao, Yanping; Chen, Xuelian; Xu, Liuyi

    2016-01-01

    Pinus massoniaia Lamb has gained more and more attention as the most important tree species for timber and forestation in South China. Gene expression studies are of great importance to identify new and elite cultivars. Real-time quantitative PCR, a highly sensitive and specific method, is commonly used in the analysis of gene expression. The appropriate reference genes must be employed to normalize the calculation program for ascertaining repeatable and significant results. Herein, eleven housekeeping genes were evaluated during different stages of P. massoniana post nematode inoculation in this study. Three statistical approaches such as geNorm, NormFinder and BestKeeper were selected to analyze the stability of candidate genes. The results indicated that U2af and β-TUB were the most stable reference genes. These two genes could be used for the normalization in most of the experiments of P. massoniana, while Histone and AK were the least stable ones. In addition, EF expressed at the lowest average Ct value was the most abundant candidate gene. As an important gene associated with defense mechanisms, ABC transporter was analyzed by qRT-PCR, and the results were used to confirm the reliability of two genes. The selected reference genes in the present study will be conducive to future gene expression normalized by qRT-PCR in P. massoniana.

  13. Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies.

    PubMed

    Kosinová, Lucie; Cahová, Monika; Fábryová, Eva; Týcová, Irena; Koblas, Tomáš; Leontovyč, Ivan; Saudek, František; Kříž, Jan

    2016-01-01

    The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards.

  14. Contamination of potable water by enterotoxigenic Escherichia coli: qPCR based culture-free detection and quantification.

    PubMed

    Patel, C B; Vajpayee, P; Singh, G; Upadhyay, R S; Shanker, R

    2011-11-01

    Tourists visiting to endemic zones may acquire Enterotoxigenic Escherichia coli (ETEC) infection resulting into diarrhea due to consumption of contaminated potable waters. In this study, a qPCR assay (SYBR Green), targeting LT1 and ST1 genes was designed to quantify ETEC in potable waters derived from civic water supply. The assay could detect lowest 1CFU/PCR targeting LT1/ST1 gene from ten-fold diluted culture of the reference strain (E. coli MTCC 723) and is ten-fold more sensitive than the conventional PCR. The quantification of the ETEC in potable waters collected from civic supply of a major city of the northern India exhibiting high flow of tourists reveals that all the sites that ran along sewage line were contaminated by the ETEC. Contamination was due to percolation of sewage. The assay could be used for the regular monitoring of potable water in places exhibiting heavy flow of tourists to prevent ETEC induced diarrhea.

  15. Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in Stipa grandis during Environmental Stresses

    PubMed Central

    Yang, Qi; Zou, Bo; Ren, Weibo; Ding, Yong; Wang, Zhen; Wang, Ruigang; Wang, Kai; Hou, Xiangyang

    2017-01-01

    Stipa grandis P. Smirn. is a dominant plant species in the typical steppe of the Xilingole Plateau of Inner Mongolia. Selection of suitable reference genes for the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is important for gene expression analysis and research into the molecular mechanisms underlying the stress responses of S. grandis. In the present study, 15 candidate reference genes (EF1 beta, ACT, GAPDH, SamDC, CUL4, CAP, SNF2, SKIP1, SKIP5, SKIP11, UBC2, UBC15, UBC17, UCH, and HERC2) were evaluated for their stability as potential reference genes for qRT-PCR under different stresses. Four algorithms were used: GeNorm, NormFinder, BestKeeper, and RefFinder. The results showed that the most stable reference genes were different under different stress conditions: EF1beta and UBC15 during drought and salt stresses; ACT and GAPDH under heat stress; SKIP5 and UBC17 under cold stress; UBC15 and HERC2 under high pH stress; UBC2 and UBC15 under wounding stress; EF1beta and UBC17 under jasmonic acid treatment; UBC15 and CUL4 under abscisic acid treatment; and HERC2 and UBC17 under salicylic acid treatment. EF1beta and HERC2 were the most suitable genes for the global analysis of all samples. Furthermore, six target genes, SgPOD, SgPAL, SgLEA, SgLOX, SgHSP90 and SgPR1, were selected to validate the most and least stable reference genes under different treatments. Our results provide guidelines for reference gene selection for more accurate qRT-PCR quantification and will promote studies of gene expression in S. grandis subjected to environmental stress. PMID:28056110

  16. Reference genes selection for transcript normalization in kenaf (Hibiscus cannabinus L.) under salinity and drought stress.

    PubMed

    Niu, Xiaoping; Qi, Jianmin; Chen, Meixia; Zhang, Gaoyang; Tao, Aifen; Fang, Pingping; Xu, Jiantang; Onyedinma, Sandra A; Su, Jianguang

    2015-01-01

    Kenaf (Hibiscus cannabinus) is an economic and ecological fiber crop but suffers severe losses in fiber yield and quality under the stressful conditions of excess salinity and drought. To explore the mechanisms by which kenaf responds to excess salinity and drought, gene expression was performed at the transcriptomic level using RNA-seq. Thus, it is crucial to have a suitable set of reference genes to normalize target gene expression in kenaf under different conditions using real-time quantitative reverse transcription-PCR (qRT-PCR). In this study, we selected 10 candidate reference genes from the kenaf transcriptome and assessed their expression stabilities by qRT-PCR in 14 NaCl- and PEG-treated samples using geNorm, NormFinder, and BestKeeper. The results indicated that TUBα and 18S rRNA were the optimum reference genes under conditions of excess salinity and drought in kenaf. Moreover, TUBα and 18S rRNA were used singly or in combination as reference genes to validate the expression levels of WRKY28 and WRKY32 in NaCl- and PEG-treated samples by qRT-PCR. The results further proved the reliability of the two selected reference genes. This work will benefit future studies on gene expression and lead to a better understanding of responses to excess salinity and drought in kenaf.

  17. Reference genes selection for transcript normalization in kenaf (Hibiscus cannabinus L.) under salinity and drought stress

    PubMed Central

    Niu, Xiaoping; Chen, Meixia; Zhang, Gaoyang; Tao, Aifen; Fang, Pingping; Xu, Jiantang; Onyedinma, Sandra A.

    2015-01-01

    Kenaf (Hibiscus cannabinus) is an economic and ecological fiber crop but suffers severe losses in fiber yield and quality under the stressful conditions of excess salinity and drought. To explore the mechanisms by which kenaf responds to excess salinity and drought, gene expression was performed at the transcriptomic level using RNA-seq. Thus, it is crucial to have a suitable set of reference genes to normalize target gene expression in kenaf under different conditions using real-time quantitative reverse transcription-PCR (qRT-PCR). In this study, we selected 10 candidate reference genes from the kenaf transcriptome and assessed their expression stabilities by qRT-PCR in 14 NaCl- and PEG-treated samples using geNorm, NormFinder, and BestKeeper. The results indicated that TUBα and 18S rRNA were the optimum reference genes under conditions of excess salinity and drought in kenaf. Moreover, TUBα and 18S rRNA were used singly or in combination as reference genes to validate the expression levels of WRKY28 and WRKY32 in NaCl- and PEG-treated samples by qRT-PCR. The results further proved the reliability of the two selected reference genes. This work will benefit future studies on gene expression and lead to a better understanding of responses to excess salinity and drought in kenaf. PMID:26644967

  18. Identification and Evaluation of Reference Genes for Quantitative Analysis of Brazilian Pine (Araucaria angustifolia Bertol. Kuntze) Gene Expression.

    PubMed

    Elbl, Paula; Navarro, Bruno V; de Oliveira, Leandro F; Almeida, Juliana; Mosini, Amanda C; Dos Santos, André L W; Rossi, Magdalena; Floh, Eny I S

    2015-01-01

    Quantitative analysis of gene expression is a fundamental experimental approach in many fields of plant biology, but it requires the use of internal controls representing constitutively expressed genes for reliable transcript quantification. In this study, we identified fifteen putative reference genes from an A. angustifolia transcriptome database. Variation in transcript levels was first evaluated in silico by comparing read counts and then by quantitative real-time PCR (qRT-PCR), resulting in the identification of six candidate genes. The consistency of transcript abundance was also calculated applying geNorm and NormFinder software packages followed by a validation approach using four target genes. The results presented here indicate that a diverse set of samples should ideally be used in order to identify constitutively expressed genes, and that the use of any two reference genes in combination, of the six tested genes, is sufficient for effective expression normalization. Finally, in agreement with the in silico prediction, a comprehensive analysis of the qRT-PCR data combined with validation analysis revealed that AaEIF4B-L and AaPP2A are the most suitable reference genes for comparative studies of A. angustifolia gene expression.

  19. Identification of stable reference genes in differentiating human pluripotent stem cells.

    PubMed

    Holmgren, Gustav; Ghosheh, Nidal; Zeng, Xianmin; Bogestål, Yalda; Sartipy, Peter; Synnergren, Jane

    2015-06-01

    Reference genes, often referred to as housekeeping genes (HKGs), are frequently used to normalize gene expression data based on the assumption that they are expressed at a constant level in the cells. However, several studies have shown that there may be a large variability in the gene expression levels of HKGs in various cell types. In a previous study, employing human embryonic stem cells (hESCs) subjected to spontaneous differentiation, we observed that the expression of commonly used HKG varied to a degree that rendered them inappropriate to use as reference genes under those experimental settings. Here we present a substantially extended study of the HKG signature in human pluripotent stem cells (hPSC), including nine global gene expression datasets from both hESC and human induced pluripotent stem cells, obtained during directed differentiation toward endoderm-, mesoderm-, and ectoderm derivatives. Sets of stably expressed genes were compiled, and a handful of genes (e.g., EID2, ZNF324B, CAPN10, and RABEP2) were identified as generally applicable reference genes in hPSCs across all cell lines and experimental conditions. The stability in gene expression profiles was confirmed by reverse transcription quantitative PCR analysis. Taken together, the current results suggest that differentiating hPSCs have a distinct HKG signature, which in some aspects is different from somatic cell types, and underscore the necessity to validate the stability of reference genes under the actual experimental setup used. In addition, the novel putative HKGs identified in this study can preferentially be used for normalization of gene expression data obtained from differentiating hPSCs.

  20. An efficient RNA isolation procedure and identification of reference genes for normalization of gene expression in blueberry.

    PubMed

    Vashisth, Tripti; Johnson, Lisa Klima; Malladi, Anish

    2011-12-01

    Application of transcriptomics approaches can greatly enhance our understanding of blueberry physiology. The success of transcriptomics approaches is dependent on the extraction of high-quality RNA which is complicated by the abundance of polyphenolics and polysaccharides in blueberry. Additionally, transcriptomics requires the accurate quantification of transcript abundance. Quantitative real-time polymerase chain reaction (qRT-PCR) is a robust method to determine transcript abundance. Normalization of gene expression using stably expressed reference genes is essential in qRT-PCR. An evaluation of the stability of expression of reference genes has not yet been reported in blueberry. The objectives of this study were to develop an effective procedure for extracting RNA from different organs and to evaluate potential reference genes for qRT-PCR analyses in blueberry. RNA of high quality and yield was extracted from eight and six organs of rabbiteye and southern highbush blueberry, respectively, using a modified cetyltrimethyl ammonium bromide-based method. The expression stability of 12 reference genes was evaluated. UBIQUITIN-CONJUGATING ENZYME (UBC28), RNA HELICASE-LIKE (RH8), CLATHRIN ADAPTER COMPLEXES MEDIUM SUBUNIT FAMILY PROTEIN (CACSa), and POLYUBIQUITIN (UBQ3b) were the most stably expressed genes across multiple organs in both blueberry species. Further, the expression stability of the reference genes in the branch abscission zone following treatment with fruit abscission-inducing compounds was analyzed. CACSa, RH8, and UBC28 were the most stably expressed genes in the abscission zone under abscission-inducing conditions. We suggest a preliminary evaluation of UBC28, CACSa, RH8, and UBQ3b to identify the most suitable reference genes for the experimental conditions under consideration in blueberry.

  1. Reference genes selection and normalization of oxidative stress responsive genes upon different temperature stress conditions in Hypericum perforatum L.

    PubMed

    Velada, Isabel; Ragonezi, Carla; Arnholdt-Schmitt, Birgit; Cardoso, Hélia

    2014-01-01

    Reverse transcription-quantitative real-time PCR (RT-qPCR) is a widely used technique for gene expression analysis. The reliability of this method depends largely on the suitable selection of stable reference genes for accurate data normalization. Hypericum perforatum L. (St. John's wort) is a field growing plant that is frequently exposed to a variety of adverse environmental stresses that can negatively affect its productivity. This widely known medicinal plant with broad pharmacological properties (anti-depressant, anti-tumor, anti-inflammatory, antiviral, antioxidant, anti-cancer, and antibacterial) has been overlooked with respect to the identification of reference genes suitable for RT-qPCR data normalization. In this study, 11 candidate reference genes were analyzed in H. perforatum plants subjected to cold and heat stresses. The expression stability of these genes was assessed using GeNorm, NormFinder and BestKeeper algorithms. The results revealed that the ranking of stability among the three algorithms showed only minor differences within each treatment. The best-ranked reference genes differed between cold- and heat-treated samples; nevertheless, TUB was the most stable gene in both experimental conditions. GSA and GAPDH were found to be reliable reference genes in cold-treated samples, while GAPDH showed low expression stability in heat-treated samples. 26SrRNA and H2A had the highest stabilities in the heat assay, whereas H2A was less stable in the cold assay. Finally, AOX1, AOX2, CAT1 and CHS genes, associated with plant stress responses and oxidative stress, were used as target genes to validate the reliability of identified reference genes. These target genes showed differential expression profiles over time in treated samples. This study not only is the first systematic analysis for the selection of suitable reference genes for RT-qPCR studies in H. perforatum subjected to temperature stress conditions, but may also provide valuable information

  2. Reference Genes Selection and Normalization of Oxidative Stress Responsive Genes upon Different Temperature Stress Conditions in Hypericum perforatum L

    PubMed Central

    Velada, Isabel; Ragonezi, Carla; Arnholdt-Schmitt, Birgit; Cardoso, Hélia

    2014-01-01

    Reverse transcription-quantitative real-time PCR (RT-qPCR) is a widely used technique for gene expression analysis. The reliability of this method depends largely on the suitable selection of stable reference genes for accurate data normalization. Hypericum perforatum L. (St. John's wort) is a field growing plant that is frequently exposed to a variety of adverse environmental stresses that can negatively affect its productivity. This widely known medicinal plant with broad pharmacological properties (anti-depressant, anti-tumor, anti-inflammatory, antiviral, antioxidant, anti-cancer, and antibacterial) has been overlooked with respect to the identification of reference genes suitable for RT-qPCR data normalization. In this study, 11 candidate reference genes were analyzed in H. perforatum plants subjected to cold and heat stresses. The expression stability of these genes was assessed using GeNorm, NormFinder and BestKeeper algorithms. The results revealed that the ranking of stability among the three algorithms showed only minor differences within each treatment. The best-ranked reference genes differed between cold- and heat-treated samples; nevertheless, TUB was the most stable gene in both experimental conditions. GSA and GAPDH were found to be reliable reference genes in cold-treated samples, while GAPDH showed low expression stability in heat-treated samples. 26SrRNA and H2A had the highest stabilities in the heat assay, whereas H2A was less stable in the cold assay. Finally, AOX1, AOX2, CAT1 and CHS genes, associated with plant stress responses and oxidative stress, were used as target genes to validate the reliability of identified reference genes. These target genes showed differential expression profiles over time in treated samples. This study not only is the first systematic analysis for the selection of suitable reference genes for RT-qPCR studies in H. perforatum subjected to temperature stress conditions, but may also provide valuable information

  3. New in-depth rainbow trout transcriptome reference and digital atlas of gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sequencing the rainbow trout genome is underway and a transcriptome reference sequence is required to help in genome assembly and gene discovery. Previously, we reported a transcriptome reference sequence using a 19X coverage of 454-pyrosequencing data. Although this work added a great wealth of ann...

  4. Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR

    PubMed Central

    Ling, Hui; Wu, Qibin; Guo, Jinlong; Xu, Liping; Que, Youxiong

    2014-01-01

    The increasingly used real time quantitative reverse transcription-PCR (qRT-PCR) method for gene expression analysis requires one or several reference gene(s) acting as normalization factor(s). In order to facilitate gene expression studies in sugarcane (Saccharum officinarum), a non-model plant with limited genome information, the stability of 13 candidate reference genes was evaluated. The geNorm, NormFinder and deltaCt methods were used for selecting stably expressed internal controls across different tissues and under various experimental treatments. These results revealed that, among these 13 candidate reference genes, GAPDH, eEF-1a and eIF-4α were the most stable and suitable for use as normalization factors across all various experimental samples. In addition, APRT could be a candidate for examining the relationship between gene copy number and transcript levels in sugarcane tissue samples. According to the results evaluated by geNorm, combining CUL and eEF-1α in hormone treatment experiments; CAC and CUL in abiotic stress tests; GAPDH, eEF-1a and CUL in all treatment samples plus CAC, CUL, APRT and TIPS-41 in cultivar tissues as groups for normalization would lead to more accurate and reliable expression quantification in sugarcane. This is the first systematic validation of reference genes for quantification of transcript expression profiles in sugarcane. This study should provide useful information for selecting reference genes for more accurate quantification of gene expression in sugarcane and other plant species. PMID:24823940

  5. Effect of carbon monoxide on gene expression in cerebrocortical astrocytes: Validation of reference genes for quantitative real-time PCR.

    PubMed

    Oliveira, Sara R; Vieira, Helena L A; Duarte, Carlos B

    2015-09-15

    Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a widely used technique to characterize changes in gene expression in complex cellular and tissue processes, such as cytoprotection or inflammation. The accurate assessment of changes in gene expression depends on the selection of adequate internal reference gene(s). Carbon monoxide (CO) affects several metabolic pathways and de novo protein synthesis is crucial in the cellular responses to this gasotransmitter. Herein a selection of commonly used reference genes was analyzed to identify the most suitable internal control genes to evaluate the effect of CO on gene expression in cultured cerebrocortical astrocytes. The cells were exposed to CO by treatment with CORM-A1 (CO releasing molecule A1) and four different algorithms (geNorm, NormFinder, Delta Ct and BestKeeper) were applied to evaluate the stability of eight putative reference genes. Our results indicate that Gapdh (glyceraldehyde-3-phosphate dehydrogenase) together with Ppia (peptidylpropyl isomerase A) is the most suitable gene pair for normalization of qRT-PCR results under the experimental conditions used. Pgk1 (phosphoglycerate kinase 1), Hprt1 (hypoxanthine guanine phosphoribosyl transferase I), Sdha (Succinate Dehydrogenase Complex, Subunit A), Tbp (TATA box binding protein), Actg1 (actin gamma 1) and Rn18s (18S rRNA) genes presented less stable expression profiles in cultured cortical astrocytes exposed to CORM-A1 for up to 60 min. For validation, we analyzed the effect of CO on the expression of Bdnf and bcl-2. Different results were obtained, depending on the reference genes used. A significant increase in the expression of both genes was found when the results were normalized with Gapdh and Ppia, in contrast with the results obtained when the other genes were used as reference. These findings highlight the need for a proper and accurate selection of the reference genes used in the quantification of qRT-PCR results

  6. Optimizing stem-loop qPCR assays through multiplexed cDNA synthesis of U6 and miRNAs

    PubMed Central

    Turner, Marie; Adhikari, Sajag; Subramanian, Senthil

    2013-01-01

    We recently reported that hairpin (or stem-loop) priming is better-suited than polyA tailing to generate cDNA for plant microRNA qPCR. One major limitation of this method is the need to perform individual cDNA synthesis reactions for the reference gene and test miRNAs. Here, we report a novel fusion primer that allows multiplexed hairpin cDNA synthesis of the most-commonly used reference gene, nucleolar small RNA U6, together with test miRNAs. We also propose the use of miR1515 as a house keeping control for tropical legumes. We show that multiplexed cDNA synthesis does not result in loss of sensitivity and reduces the amount of RNA required for miRNA gene expression assays. PMID:23673353

  7. Identification of suitable reference genes for quantitative gene expression analysis in rat adipose stromal cells induced to trilineage differentiation.

    PubMed

    Santos, Bruno Paiva Dos; da Costa Diesel, Luciana Fraga; da Silva Meirelles, Lindolfo; Nardi, Nance Beyer; Camassola, Melissa

    2016-12-15

    This study was designed to (i) identify stable reference genes for the analysis of gene expression during in vitro differentiation of rat adipose stromal cells (rASCs), (ii) recommend stable genes for individual treatment conditions, and (iii) validate these genes by comparison with normalization results from stable and unstable reference genes. On the basis of a literature review, eight genes were selected: Actb, B2m, Hprt1, Ppia, Rplp0, Rpl13a, Rpl5, and Ywhaz. Genes were ranked according to their stability under different culture conditions as assessed using GenNorm, NormFinder, and RefFinder algorithms. Although the employed algorithms returned different rankings, the most frequently top-ranked genes were: B2m and/or Ppia for all 28day treatments (ALL28); Ppia and Hprt1 (adipogenic differentiation; A28), B2m (chondrogenic differentiation; C28), Rpl5 (controls maintained in complete culture medium; CCM), Rplp0 (osteogenic differentiation for 3days; O3), Rpl13a and Actb (osteogenic differentiation for 7days; O7), Rplp0 and Ppia (osteogenic differentiation for 14days; O14), Hprt1 and Ppia (osteogenic differentiation for 28days; O28), as well as Actb (all osteogenesis time points combined; ALLOSTEO). The obtained results indicate that the performance of reference genes depends on the differentiation protocol and on the analysis time, thus providing valuable information for the design of RT-PCR experiments.

  8. Evaluation and validation of reference gene stability during Marek's disease virus (MDV) infection.

    PubMed

    Neerukonda, Sabari Nath; Katneni, Upendra K; Golovan, Sergey; Parcells, Mark S

    2016-10-01

    Quantitative RT-PCR (qRT-PCR) is widely used in the study of relative gene expression in general, and has been used in the field of Marek's disease (MD) research to measure transcriptional responses to infection and/or vaccination. Studies in the past have either employed cellular β-actin (BACT) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal reference genes, although the stability of their expression in the context of Marek's disease virus (MDV) infection has never been investigated. In the present study, we compared the stability of five reference genes (BACT, 28S RNA, 18S RNA, GAPDH, Peptidyl-prolyl-isomerase B [PPIB], a.k.a. cyclophilin B) as standard internal controls in chicken embryo fibroblast (CEFs) cultures infected with either MD vaccine or oncogenic MDV1 viruses. We further extend these analyses to reference gene stability in spleen lymphomas induced by infection of commercial broiler chickens with a very virulent plus MDV1 (vv+ TK-2a virus). Two excel based algorithms, (Bestkeeper and Normfinder) were employed to compare reference gene stability. Bestkeeper and Normfinder analysis of reference gene stability in virus- and mock-infected cells, showed that 28S RNA and PPIB displayed higher stability in CEF infections with either oncogenic or vaccine viruses. In addition, both Bestkeeper and Normfinder determined 28S RNA and PPIB to be the most stably-expressed reference genes in vivo in vv+ TK-2a-induced spleen lymphomas. Furthermore, Bestkeeper and Normfinder analyses both determined BACT to be the least stable reference gene during MDV infection of CEF with oncogenic viruses, vaccine viruses, as well as in vv+ TK-2a-induced spleen lymphomas.

  9. METHOD TO CLASSIFY ENVIRONMENTAL SAMPLES BASED ON MOLD ANALYSES BY QPCR

    EPA Science Inventory

    A total of 82 quantitative PCR (QPCR) assays were used to identify and quantify different indoor molds in dust samples from the homes of six infants suffering from pulmonary hemorrhage and 26 reference homes in Cleveland, Ohio. No significant difference was seen in the total cell...

  10. Reference gene selection for quantitative real-time PCR normalization in Quercus suber.

    PubMed

    Marum, Liliana; Miguel, Andreia; Ricardo, Cândido P; Miguel, Célia

    2012-01-01

    The use of reverse transcription quantitative PCR technology to assess gene expression levels requires an accurate normalization of data in order to avoid misinterpretation of experimental results and erroneous analyses. Despite being the focus of several transcriptomics projects, oaks, and particularly cork oak (Quercus suber), have not been investigated regarding the identification of reference genes suitable for the normalization of real-time quantitative PCR data. In this study, ten candidate reference genes (Act, CACs, EF-1α, GAPDH, His3, PsaH, Sand, PP2A, ß-Tub and Ubq) were evaluated to determine the most stable internal reference for quantitative PCR normalization in cork oak. The transcript abundance of these genes was analysed in several tissues of cork oak, including leaves, reproduction cork, and periderm from branches at different developmental stages (1-, 2-, and 3-year old) or collected in different dates (active growth period versus dormancy). The three statistical methods (geNorm, NormFinder, and CV method) used in the evaluation of the most suitable combination of reference genes identified Act and CACs as the most stable candidates when all the samples were analysed together, while ß-Tub and PsaH showed the lowest expression stability. However, when different tissues, developmental stages, and collection dates were analysed separately, the reference genes exhibited some variation in their expression levels. In this study, and for the first time, we have identified and validated reference genes in cork oak that can be used for quantification of target gene expression in different tissues and experimental conditions and will be useful as a starting point for gene expression studies in other oaks.

  11. Sampling and Pooling Methods for Capturing Herd Level Antibiotic Resistance in Swine Feces using qPCR and CFU Approaches.

    PubMed

    Schmidt, Gunilla Veslemøy; Mellerup, Anders; Christiansen, Lasse Engbo; Ståhl, Marie; Olsen, John Elmerdahl; Angen, Øystein

    2015-01-01

    The aim of this article was to define the sampling level and method combination that captures antibiotic resistance at pig herd level utilizing qPCR antibiotic resistance gene quantification and culture-based quantification of antibiotic resistant coliform indicator bacteria. Fourteen qPCR assays for commonly detected antibiotic resistance genes were developed, and used to quantify antibiotic resistance genes in total DNA from swine fecal samples that were obtained using different sampling and pooling methods. In parallel, the number of antibiotic resistant coliform indicator bacteria was determined in the same swine fecal samples. The results showed that the qPCR assays were capable of detecting differences in antibiotic resistance levels in individual animals that the coliform bacteria colony forming units (CFU) could not. Also, the qPCR assays more accurately quantified antibiotic resistance genes when comparing individual sampling and pooling methods. qPCR on pooled samples was found to be a good representative for the general resistance level in a pig herd compared to the coliform CFU counts. It had significantly reduced relative standard deviations compared to coliform CFU counts in the same samples, and therefore differences in antibiotic resistance levels between samples were more readily detected. To our knowledge, this is the first study to describe sampling and pooling methods for qPCR quantification of antibiotic resistance genes in total DNA extracted from swine feces.

  12. Identification of Reference Genes for Quantitative Gene Expression Studies in a Non-Model Tree Pistachio (Pistacia vera L.).

    PubMed

    Moazzam Jazi, Maryam; Ghadirzadeh Khorzoghi, Effat; Botanga, Christopher; Seyedi, Seyed Mahdi

    2016-01-01

    The tree species, Pistacia vera (P. vera) is an important commercial product that is salt-tolerant and long-lived, with a possible lifespan of over one thousand years. Gene expression analysis is an efficient method to explore the possible regulatory mechanisms underlying these characteristics. Therefore, having the most suitable set of reference genes is required for transcript level normalization under different conditions in P. vera. In the present study, we selected eight widely used reference genes, ACT, EF1α, α-TUB, β-TUB, GAPDH, CYP2, UBQ10, and 18S rRNA. Using qRT-PCR their expression was assessed in 54 different samples of three cultivars of P. vera. The samples were collected from different organs under various abiotic treatments (cold, drought, and salt) across three time points. Several statistical programs (geNorm, NormFinder, and BestKeeper) were applied to estimate the expression stability of candidate reference genes. Results obtained from the statistical analysis were then exposed to Rank aggregation package to generate a consensus gene rank. Based on our results, EF1α was found to be the superior reference gene in all samples under all abiotic treatments. In addition to EF1α, ACT and β-TUB were the second best reference genes for gene expression analysis in leaf and root. We recommended β-TUB as the second most stable gene for samples under the cold and drought treatments, while ACT holds the same position in samples analyzed under salt treatment. This report will benefit future research on the expression profiling of P. vera and other members of the Anacardiaceae family.

  13. Identification of Reference Genes for Quantitative Gene Expression Studies in a Non-Model Tree Pistachio (Pistacia vera L.)

    PubMed Central

    Moazzam Jazi, Maryam; Ghadirzadeh Khorzoghi, Effat; Botanga, Christopher; Seyedi, Seyed Mahdi

    2016-01-01

    The tree species, Pistacia vera (P. vera) is an important commercial product that is salt-tolerant and long-lived, with a possible lifespan of over one thousand years. Gene expression analysis is an efficient method to explore the possible regulatory mechanisms underlying these characteristics. Therefore, having the most suitable set of reference genes is required for transcript level normalization under different conditions in P. vera. In the present study, we selected eight widely used reference genes, ACT, EF1α, α-TUB, β-TUB, GAPDH, CYP2, UBQ10, and 18S rRNA. Using qRT-PCR their expression was assessed in 54 different samples of three cultivars of P. vera. The samples were collected from different organs under various abiotic treatments (cold, drought, and salt) across three time points. Several statistical programs (geNorm, NormFinder, and BestKeeper) were applied to estimate the expression stability of candidate reference genes. Results obtained from the statistical analysis were then exposed to Rank aggregation package to generate a consensus gene rank. Based on our results, EF1α was found to be the superior reference gene in all samples under all abiotic treatments. In addition to EF1α, ACT and β-TUB were the second best reference genes for gene expression analysis in leaf and root. We recommended β-TUB as the second most stable gene for samples under the cold and drought treatments, while ACT holds the same position in samples analyzed under salt treatment. This report will benefit future research on the expression profiling of P. vera and other members of the Anacardiaceae family. PMID:27308855

  14. Reference gene validation for quantitative RT-PCR during biotic and abiotic stresses in Vitis vinifera.

    PubMed

    Borges, Alexandre Filipe; Fonseca, Catarina; Ferreira, Ricardo Boavida; Lourenço, Ana Maria; Monteiro, Sara

    2014-01-01

    Grapevine is one of the most cultivated fruit crop worldwide with Vitis vinifera being the species with the highest economical importance. Being highly susceptible to fungal pathogens and increasingly affected by environmental factors, it has become an important agricultural research area, where gene expression analysis plays a fundamental role. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is currently amongst the most powerful techniques to perform gene expression studies. Nevertheless, accurate gene expression quantification strongly relies on appropriate reference gene selection for sample normalization. Concerning V. vinifera, limited information still exists as for which genes are the most suitable to be used as reference under particular experimental conditions. In this work, seven candidate genes were investigated for their stability in grapevine samples referring to four distinct stresses (Erysiphe necator, wounding and UV-C irradiation in leaves and Phaeomoniella chlamydospora colonization in wood). The expression stability was evaluated using geNorm, NormFinder and BestKeeper. In all cases, full agreement was not observed for the three methods. To provide comprehensive rankings integrating the three different programs, for each treatment, a consensus ranking was created using a non-weighted unsupervised rank aggregation method. According to the last, the three most suitable reference genes to be used in grapevine leaves, regardless of the stress, are UBC, VAG and PEP. For the P. chlamydospora treatment, EF1, CYP and UBC were the best scoring genes. Acquaintance of the most suitable reference genes to be used in grapevine samples can contribute for accurate gene expression quantification in forthcoming studies.

  15. Reference Gene Validation for Quantitative RT-PCR during Biotic and Abiotic Stresses in Vitis vinifera

    PubMed Central

    Borges, Alexandre Filipe; Fonseca, Catarina; Ferreira, Ricardo Boavida; Lourenço, Ana Maria; Monteiro, Sara

    2014-01-01

    Grapevine is one of the most cultivated fruit crop worldwide with Vitis vinifera being the species with the highest economical importance. Being highly susceptible to fungal pathogens and increasingly affected by environmental factors, it has become an important agricultural research area, where gene expression analysis plays a fundamental role. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is currently amongst the most powerful techniques to perform gene expression studies. Nevertheless, accurate gene expression quantification strongly relies on appropriate reference gene selection for sample normalization. Concerning V. vinifera, limited information still exists as for which genes are the most suitable to be used as reference under particular experimental conditions. In this work, seven candidate genes were investigated for their stability in grapevine samples referring to four distinct stresses (Erysiphe necator, wounding and UV-C irradiation in leaves and Phaeomoniella chlamydospora colonization in wood). The expression stability was evaluated using geNorm, NormFinder and BestKeeper. In all cases, full agreement was not observed for the three methods. To provide comprehensive rankings integrating the three different programs, for each treatment, a consensus ranking was created using a non-weighted unsupervised rank aggregation method. According to the last, the three most suitable reference genes to be used in grapevine leaves, regardless of the stress, are UBC, VAG and PEP. For the P. chlamydospora treatment, EF1, CYP and UBC were the best scoring genes. Acquaintance of the most suitable reference genes to be used in grapevine samples can contribute for accurate gene expression quantification in forthcoming studies. PMID:25340748

  16. Reference gene selection for quantitative gene expression studies during biological invasions: A test on multiple genes and tissues in a model ascidian Ciona savignyi.

    PubMed

    Huang, Xuena; Gao, Yangchun; Jiang, Bei; Zhou, Zunchun; Zhan, Aibin

    2016-01-15

    As invasive species have successfully colonized a wide range of dramatically different local environments, they offer a good opportunity to study interactions between species and rapidly changing environments. Gene expression represents one of the primary and crucial mechanisms for rapid adaptation to local environments. Here, we aim to select reference genes for quantitative gene expression analysis based on quantitative Real-Time PCR (qRT-PCR) for a model invasive ascidian, Ciona savignyi. We analyzed the stability of ten candidate reference genes in three tissues (siphon, pharynx and intestine) under two key environmental stresses (temperature and salinity) in the marine realm based on three programs (geNorm, NormFinder and delta Ct method). Our results demonstrated only minor difference for stability rankings among the three methods. The use of different single reference gene might influence the data interpretation, while multiple reference genes could minimize possible errors. Therefore, reference gene combinations were recommended for different tissues - the optimal reference gene combination for siphon was RPS15 and RPL17 under temperature stress, and RPL17, UBQ and TubA under salinity treatment; for pharynx, TubB, TubA and RPL17 were the most stable genes under temperature stress, while TubB, TubA and UBQ were the best under salinity stress; for intestine, UBQ, RPS15 and RPL17 were the most reliable reference genes under both treatments. Our results suggest that the necessity of selection and test of reference genes for different tissues under varying environmental stresses. The results obtained here are expected to reveal mechanisms of gene expression-mediated invasion success using C. savignyi as a model species.

  17. Identification of Reference Genes for RT-qPCR Data Normalization in Cannabis sativa Stem Tissues

    PubMed Central

    Mangeot-Peter, Lauralie; Legay, Sylvain; Hausman, Jean-Francois; Esposito, Sergio; Guerriero, Gea

    2016-01-01

    Gene expression profiling via quantitative real-time PCR is a robust technique widely used in the life sciences to compare gene expression patterns in, e.g., different tissues, growth conditions, or after specific treatments. In the field of plant science, real-time PCR is the gold standard to study the dynamics of gene expression and is used to validate the results generated with high throughput techniques, e.g., RNA-Seq. An accurate relative quantification of gene expression relies on the identification of appropriate reference genes, that need to be determined for each experimental set-up used and plant tissue studied. Here, we identify suitable reference genes for expression profiling in stems of textile hemp (Cannabis sativa L.), whose tissues (isolated bast fibres and core) are characterized by remarkable differences in cell wall composition. We additionally validate the reference genes by analysing the expression of putative candidates involved in the non-oxidative phase of the pentose phosphate pathway and in the first step of the shikimate pathway. The goal is to describe the possible regulation pattern of some genes involved in the provision of the precursors needed for lignin biosynthesis in the different hemp stem tissues. The results here shown are useful to design future studies focused on gene expression analyses in hemp. PMID:27649158

  18. Defining suitable reference genes for RT-qPCR analysis on intestinal epithelial cells.

    PubMed

    Sirakov, Maria; Borra, Marco; Cambuli, Francesca Maria; Plateroti, Michelina

    2013-07-01

    The study of the mammalian intestinal epithelium concerns several aspects of cellular and molecular biology. In fact, most of these studies aim to define molecular components or mechanisms related with the control of stemness and the balance between cell proliferation and differentiation in physiopathological conditions. It is worth mentioning that real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) approaches are commonly used, but only a few studies are available regarding suitable reference genes to normalize gene expression data. The present study was designed to validate potential reference genes in freshly isolated proliferating or differentiated epithelial cells from the mouse intestine. We also extended our analysis to the IEC6 intestinal epithelial cells, as a promising model to study intestinal physiopathology in vitro. The stability of six potential reference genes (Hprt1, Ppia, Gapdh, Rplp0, Ppib, and Vil1) has been tested both in epithelial cells isolated from the mouse intestine and in the IEC6 cell line. The software programs-geNorm and Normfinder-were used to obtain an estimation of the expression stability of each gene and, by comparing the results, to identify the most suitable genes for RT-qPCR data normalization. These multiple approaches allowed us to select different suitable reference genes for the correct quantification of mRNAs depending on the differentiated or proliferative nature of the cells.

  19. Impact of thawing on reference gene expression stability in renal cell carcinoma samples.

    PubMed

    Ma, Yi; Dai, HuiLi; Kong, XianMing; Wang, LiMin

    2012-09-01

    More and more samples are obtained from biobanks for biomedical research; however, some of these samples may undergo thawing before processing. We aim to evaluate the reference gene expression stability in thawed renal cell carcinoma samples. Sixteen matched malignant and nonmalignant renal tissue samples were obtained and each sample was divided into 4 aliquots before being snap frozen and stored at -80°C. By quantitative real-time polymerase chain reaction, a time-course study was conducted on the thawed tissue to evaluate the expression stability of a panel of the 10 most frequently used reference genes in renal cell carcinom samples: ACTB, ALAS1, B2M, GAPDH, HMBS, HPRT, PPIA, RPLP0,TBP, and TUBB. As shown by geNorm M values, PPIA was the most stable gene at the 0-, 15-, and 30-minute time points (M=0.82, 0.85, and 0.76, respectively), whereas GAPDH was ranked last at the 5-, 15-, and 30-minute time points (M=1.38, 1.44, and 1.39, respectively). A positive correlation was found by linear regression between the thawing time and 2 to the power of crossing point values of all candidate reference genes (P<0.05). The mean coefficient of variance of all reference genes increased significantly at time points 5, 15, and 30 minutes compared with 0 minutes (P<0.01). In conclusion, using the geNorm algorithm, PPIA was identified as the most stably expressed gene between malignant and nonmalignant renal tissue samples that were thawed for similar time periods. All the reference genes showed high variations along with the thawing time; it should be recommended to use a combination of several candidate reference genes when comparing samples thawed for different time periods.

  20. Selection of Reference Genes for Transcriptional Analysis of Edible Tubers of Potato (Solanum tuberosum L.)

    PubMed Central

    Voorhuijzen, Marleen M.; Staats, Martijn; Hutten, Ronald C. B.; Van Dijk, Jeroen P.; Kok, Esther; Frazzon, Jeverson

    2015-01-01

    Potato (Solanum tuberosum) yield has increased dramatically over the last 50 years and this has been achieved by a combination of improved agronomy and biotechnology efforts. Gene studies are taking place to improve new qualities and develop new cultivars. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a bench-marking analytical tool for gene expression analysis, but its accuracy is highly dependent on a reliable normalization strategy of an invariant reference genes. For this reason, the goal of this work was to select and validate reference genes for transcriptional analysis of edible tubers of potato. To do so, RT-qPCR primers were designed for ten genes with relatively stable expression in potato tubers as observed in RNA-Seq experiments. Primers were designed across exon boundaries to avoid genomic DNA contamination. Differences were observed in the ranking of candidate genes identified by geNorm, NormFinder and BestKeeper algorithms. The ranks determined by geNorm and NormFinder were very similar and for all samples the most stable candidates were C2, exocyst complex component sec3 (SEC3) and ATCUL3/ATCUL3A/CUL3/CUL3A (CUL3A). According to BestKeeper, the importin alpha and ubiquitin-associated/ts-n genes were the most stable. Three genes were selected as reference genes for potato edible tubers in RT-qPCR studies. The first one, called C2, was selected in common by NormFinder and geNorm, the second one is SEC3, selected by NormFinder, and the third one is CUL3A, selected by geNorm. Appropriate reference genes identified in this work will help to improve the accuracy of gene expression quantification analyses by taking into account differences that may be observed in RNA quality or reverse transcription efficiency across the samples. PMID:25830330

  1. Selection of reference genes for transcriptional analysis of edible tubers of potato (Solanum tuberosum L.).

    PubMed

    Mariot, Roberta Fogliatto; de Oliveira, Luisa Abruzzi; Voorhuijzen, Marleen M; Staats, Martijn; Hutten, Ronald C B; Van Dijk, Jeroen P; Kok, Esther; Frazzon, Jeverson

    2015-01-01

    Potato (Solanum tuberosum) yield has increased dramatically over the last 50 years and this has been achieved by a combination of improved agronomy and biotechnology efforts. Gene studies are taking place to improve new qualities and develop new cultivars. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a bench-marking analytical tool for gene expression analysis, but its accuracy is highly dependent on a reliable normalization strategy of an invariant reference genes. For this reason, the goal of this work was to select and validate reference genes for transcriptional analysis of edible tubers of potato. To do so, RT-qPCR primers were designed for ten genes with relatively stable expression in potato tubers as observed in RNA-Seq experiments. Primers were designed across exon boundaries to avoid genomic DNA contamination. Differences were observed in the ranking of candidate genes identified by geNorm, NormFinder and BestKeeper algorithms. The ranks determined by geNorm and NormFinder were very similar and for all samples the most stable candidates were C2, exocyst complex component sec3 (SEC3) and ATCUL3/ATCUL3A/CUL3/CUL3A (CUL3A). According to BestKeeper, the importin alpha and ubiquitin-associated/ts-n genes were the most stable. Three genes were selected as reference genes for potato edible tubers in RT-qPCR studies. The first one, called C2, was selected in common by NormFinder and geNorm, the second one is SEC3, selected by NormFinder, and the third one is CUL3A, selected by geNorm. Appropriate reference genes identified in this work will help to improve the accuracy of gene expression quantification analyses by taking into account differences that may be observed in RNA quality or reverse transcription efficiency across the samples.

  2. Evaluation and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) Under Drought Stress Conditions

    PubMed Central

    Sinha, Pallavi; Singh, Vikas K.; Suryanarayana, V.; Krishnamurthy, L.; Saxena, Rachit K.; Varshney, Rajeev K.

    2015-01-01

    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. PMID:25849964

  3. Evaluation and validation of housekeeping genes as reference for gene expression studies in pigeonpea (Cajanus cajan) under drought stress conditions.

    PubMed

    Sinha, Pallavi; Singh, Vikas K; Suryanarayana, V; Krishnamurthy, L; Saxena, Rachit K; Varshney, Rajeev K

    2015-01-01

    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.

  4. Reference Gene Validation for RT-qPCR, a Note on Different Available Software Packages

    PubMed Central

    De Spiegelaere, Ward; Dern-Wieloch, Jutta; Weigel, Roswitha; Schumacher, Valérie; Schorle, Hubert; Nettersheim, Daniel; Bergmann, Martin; Brehm, Ralph; Kliesch, Sabine; Vandekerckhove, Linos; Fink, Cornelia

    2015-01-01

    Background An appropriate normalization strategy is crucial for data analysis from real time reverse transcription polymerase chain reactions (RT-qPCR). It is widely supported to identify and validate stable reference genes, since no single biological gene is stably expressed between cell types or within cells under different conditions. Different algorithms exist to validate optimal reference genes for normalization. Applying human cells, we here compare the three main methods to the online available RefFinder tool that integrates these algorithms along with R-based software packages which include the NormFinder and GeNorm algorithms. Results 14 candidate reference genes were assessed by RT-qPCR in two sample sets, i.e. a set of samples of human testicular tissue containing carcinoma in situ (CIS), and a set of samples from the human adult Sertoli cell line (FS1) either cultured alone or in co-culture with the seminoma like cell line (TCam-2) or with equine bone marrow derived mesenchymal stem cells (eBM-MSC). Expression stabilities of the reference genes were evaluated using geNorm, NormFinder, and BestKeeper. Similar results were obtained by the three approaches for the most and least stably expressed genes. The R-based packages NormqPCR, SLqPCR and the NormFinder for R script gave identical gene rankings. Interestingly, different outputs were obtained between the original software packages and the RefFinder tool, which is based on raw Cq values for input. When the raw data were reanalysed assuming 100% efficiency for all genes, then the outputs of the original software packages were similar to the RefFinder software, indicating that RefFinder outputs may be biased because PCR efficiencies are not taken into account. Conclusions This report shows that assay efficiency is an important parameter for reference gene validation. New software tools that incorporate these algorithms should be carefully validated prior to use. PMID:25825906

  5. Recent advances in quantitative PCR (qPCR) applications in food microbiology.

    PubMed

    Postollec, Florence; Falentin, Hélène; Pavan, Sonia; Combrisson, Jérôme; Sohier, Danièle

    2011-08-01

    Molecular methods are being increasingly applied to detect, quantify and study microbial populations in food or during food processes. Among these methods, PCR-based techniques have been the subject of considerable focus and ISO guidelines have been established for the detection of food-borne pathogens. More particularly, real-time quantitative PCR (qPCR) is considered as a method of choice for the detection and quantification of microorganisms. One of its major advantages is to be faster than conventional culture-based methods. It is also highly sensitive, specific and enables simultaneous detection of different microorganisms. Application of reverse-transcription-qPCR (RT-qPCR) to study population dynamics and activities through quantification of gene expression in food, by contrast with the use of qPCR, is just beginning. Provided that appropriate controls are included in the analyses, qPCR and RT-qPCR appear to be highly accurate and reliable for quantification of genes and gene expression. This review addresses some important technical aspects to be considered when using these techniques. Recent applications of qPCR and RT-qPCR in food microbiology are given. Some interesting applications such as risk analysis or studying the influence of industrial processes on gene expression and microbial activity are reported.

  6. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    ERIC Educational Resources Information Center

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  7. Selection and evaluation of reference genes for analysis of mouse (Mus musculus) sex-dimorphic brain development

    PubMed Central

    Cheung, Tanya T.; Weston, Mitchell K.

    2017-01-01

    The development of the brain is sex-dimorphic, and as a result so are many neurological disorders. One approach for studying sex-dimorphic brain development is to measure gene expression in biological samples using RT-qPCR. However, the accuracy and consistency of this technique relies on the reference gene(s) selected. We analyzed the expression of ten reference genes in male and female samples over three stages of brain development, using popular algorithms NormFinder, GeNorm and Bestkeeper. The top ranked reference genes at each time point were further used to quantify gene expression of three sex-dimorphic genes (Wnt10b, Xist and CYP7B1). When comparing gene expression between the sexes expression at specific time points the best reference gene combinations are: Sdha/Pgk1 at E11.5, RpL38/Sdha E12.5, and Actb/RpL37 at E15.5. When studying expression across time, the ideal reference gene(s) differs with sex. For XY samples a combination of Actb/Sdha. In contrast, when studying gene expression across developmental stage with XX samples, Sdha/Gapdh were the top reference genes. Our results identify the best combination of two reference genes when studying male and female brain development, and emphasize the importance of selecting the correct reference genes for comparisons between developmental stages. PMID:28133578

  8. Selection of reference genes for expression analyses of red-fleshed sweet orange (Citrus sinensis).

    PubMed

    Pinheiro, T T; Nishimura, D S; De Nadai, F B; Figueira, A; Latado, R R

    2015-12-28

    Red-fleshed oranges (Citrus sinensis) contain high levels of carotenoids and lycopene. The growing consumer demand for products with health benefits has increased interest in these types of Citrus cultivars as a potential source of nutraceuticals. However, little is known about the physiology of these cultivars under Brazilian conditions. Transcriptome and gene expression analyses are important tools in the breeding and management of red-fleshed sweet orange cultivars. Reverse transcription quantitative polymerase chain reaction is a method of quantifying gene expression, but various standardizations are required to obtain precise, accurate, and specific results. Among the standardizations required, the choice of suitable stable reference genes is fundamental. The objective of this study was to evaluate the stability of 11 candidate genes using various tissue and organ samples from healthy plants or leaves from citrus greening disease (Huanglongbing)-symptomatic plants of a Brazilian red-fleshed cultivar ('Sanguínea de Mombuca'), in order to select the most suitable reference gene for investigating gene expression under these conditions. geNorm and NormFinder identified genes that encoded translation initiation factor 3, ribosomal protein L35, and translation initiation factor 5A as the most stable genes under the biological conditions tested, and genes coding actin (ACT) and the subunit of the PSI reaction center subunit III were the least stable. Phosphatase, malate dehydrogenase, and ACT were the most stable genes in the leaf samples of infected plants.

  9. Identification of qRT-PCR reference genes for analysis of opioid gene expression in a hibernator.

    PubMed

    Otis, Jessica P; Ackermann, Laynez W; Denning, Gerene M; Carey, Hannah V

    2010-04-01

    Previous work has suggested that central and peripheral opioid signaling are involved in regulating torpor behavior and tissue protection associated with the hibernation phenotype. We used quantitative real-time PCR (qRT-PCR) to measure mRNA levels of opioid peptide precursors and receptors in the brain and heart of summer ground squirrels (Ictidomys tridecemlineatus) and winter hibernating squirrels in the torpid or interbout arousal states. The use of appropriate reference genes for normalization of qRT-PCR gene expression data can have profound effects on the analysis and interpretation of results. This may be particularly important when experimental subjects, such as hibernating animals, undergo significant morphological and/or functional changes during the study. Therefore, an additional goal of this study was to identify stable reference genes for use in qRT-PCR studies of the 13-lined ground squirrel. Expression levels of 10 potential reference genes were measured in the small intestine, liver, brain, and heart, and the optimal combinations of the most stable reference genes were identified by the GeNorm Excel applet. Based on this analysis, we provide recommendations for reference genes to use in each tissue that would be suitable for comparative studies among different activity states. When appropriate normalization of mRNA levels was used, there were no changes in opioid-related genes in heart among the three activity states; in brain, DOR expression was highest during torpor, lowest in interbout arousal and intermediate in summer. The results support the idea that changes in DOR expression may regulate the level of neuronal activity in brain during the annual hibernation cycle and may contribute to hibernation-associated tissue protection.

  10. Discovery of miRNAs and Their Corresponding miRNA Genes in Atlantic Cod (Gadus morhua): Use of Stable miRNAs as Reference Genes Reveals Subgroups of miRNAs That Are Highly Expressed in Particular Organs

    PubMed Central

    Andreassen, Rune; Rangnes, Fredrik; Sivertsen, Maria; Chiang, Michelle; Tran, Michelle; Worren, Merete Molton

    2016-01-01

    Background Atlantic cod (Gadus morhua) is among the economically most important species in the northern Atlantic Ocean and a model species for studying development of the immune system in vertebrates. MicroRNAs (miRNAs) are an abundant class of small RNA molecules that regulate fundamental biological processes at the post-transcriptional level. Detailed knowledge about a species miRNA repertoire is necessary to study how the miRNA transcriptome modulate gene expression. We have therefore discovered and characterized mature miRNAs and their corresponding miRNA genes in Atlantic cod. We have also performed a validation study to identify suitable reference genes for RT-qPCR analysis of miRNA expression in Atlantic cod. Finally, we utilized the newly characterized miRNA repertoire and the dedicated RT-qPCR method to reveal miRNAs that are highly expressed in certain organs. Results The discovery analysis revealed 490 mature miRNAs (401 unique sequences) along with precursor sequences and genomic location of the miRNA genes. Twenty six of these were novel miRNA genes. Validation studies ranked gmo-miR-17-1—5p or the two-gene combination gmo-miR25-3p and gmo-miR210-5p as most suitable qPCR reference genes. Analysis by RT-qPCR revealed 45 miRNAs with significantly higher expression in tissues from one or a few organs. Comparisons to other vertebrates indicate that some of these miRNAs may regulate processes like growth, lipid metabolism, immune response to microbial infections and scar damage repair. Three teleost-specific and three novel Atlantic cod miRNAs were among the differentially expressed miRNAs. Conclusions The number of known mature miRNAs was considerably increased by our identification of miRNAs and miRNA genes in Atlantic cod. This will benefit further functional studies of miRNA expression using deep sequencing methods. The validation study showed that stable miRNAs are suitable reference genes for RT-qPCR analysis of miRNA expression. Applying RT-qPCR we

  11. A brain region-specific predictive gene map for autism derived by profiling a reference gene set.

    PubMed

    Kumar, Ajay; Swanwick, Catherine Croft; Johnson, Nicole; Menashe, Idan; Basu, Saumyendra N; Bales, Michael E; Banerjee-Basu, Sharmila

    2011-01-01

    Molecular underpinnings of complex psychiatric disorders such as autism spectrum disorders (ASD) remain largely unresolved. Increasingly, structural variations in discrete chromosomal loci are implicated in ASD, expanding the search space for its disease etiology. We exploited the high genetic heterogeneity of ASD to derive a predictive map of candidate genes by an integrated bioinformatics approach. Using a reference set of 84 Rare and Syndromic candidate ASD genes (AutRef84), we built a composite reference profile based on both functional and expression analyses. First, we created a functional profile of AutRef84 by performing Gene Ontology (GO) enrichment analysis which encompassed three main areas: 1) neurogenesis/projection, 2) cell adhesion, and 3) ion channel activity. Second, we constructed an expression profile of AutRef84 by conducting DAVID analysis which found enrichment in brain regions critical for sensory information processing (olfactory bulb, occipital lobe), executive function (prefrontal cortex), and hormone secretion (pituitary). Disease specificity of this dual AutRef84 profile was demonstrated by comparative analysis with control, diabetes, and non-specific gene sets. We then screened the human genome with the dual AutRef84 profile to derive a set of 460 potential ASD candidate genes. Importantly, the power of our predictive gene map was demonstrated by capturing 18 existing ASD-associated genes which were not part of the AutRef84 input dataset. The remaining 442 genes are entirely novel putative ASD risk genes. Together, we used a composite ASD reference profile to generate a predictive map of novel ASD candidate genes which should be prioritized for future research.

  12. Exploring Valid Reference Genes for Quantitative Real-Time PCR Analysis in Sesamia inferens (Lepidoptera: Noctuidae)

    PubMed Central

    Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

    2015-01-01

    The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and β-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (−8, −6, −4, −2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens. PMID:25585250

  13. Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae).

    PubMed

    Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

    2015-01-01

    The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and β-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

  14. Evaluation of reference genes for real-time quantitative PCR in the marine flavobacterium Zobellia galactanivorans.

    PubMed

    Thomas, François; Barbeyron, Tristan; Michel, Gurvan

    2011-01-01

    The marine bacteria Zobellia galactanivorans is an emerging model microorganism for the bioconversion of algal polysaccharides. The sequence analysis of its genome opens the way to in-depth gene expression analysis, such as reverse transcription quantitative PCR (RT-qPCR) studies. The selection and validation of reference genes are a mandatory first step for the accurate quantification of transcripts. We selected fourteen candidate reference genes belonging to distinct pathways, namely replication, transcription, translation, citric acid cycle, amino acid, nucleotide and dihydrofolate metabolisms, and peptidoglycan, FMN and aromatic compounds synthesis. We quantified their expression by RT-qPCR in various culture conditions corresponding to different temperatures, carbon sources or stresses. The applications geNorm and Normfinder allowed ranking the genes according to their stability and gave concordant results. We found that the geometric average of the expression of glyA, icdA and gmkA can be confidently used to normalize the transcript abundance of genes of interest. In conclusion, this work provides a reliable procedure for gene expression analysis in the flavobacterium Z. galactanivorans and a validated set of reference genes to be used in future transcriptomics approaches. The strategy developed could also be the starting point for similar studies in other members of the Flavobacteria class.

  15. Selection of endogenous reference genes for gene expression analysis in the Mediterranean species of the Bemisia tabaci (Hemiptera: Aleyrodidae) complex.

    PubMed

    Su, Yun; He, Wen-Bo; Wang, Jia; Li, Jun-Min; Liu, Shu-Sheng; Wang, Xiao-Wei

    2013-06-01

    Quantitative real-time reverse transcription polymerase chain reaction is widely used for gene expression analysis, and robust normalization against stably expressed endogenous reference genes (ERGs) is necessary to obtain accurate results. In this study, the stability of nine housekeeping genes of the sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) Mediterranean were evaluated in various conditions by quantitative real-time reverse transcription polymerase chain reaction using geNorm and Normfinder programs. Both programs suggested alpha-tubulin/ubiquitin and 18S small subunit ribosomal RNA the most stable genes for bacterium- and insecticide-treated whiteflies, respectively. For developmental stages, organs, and the samples including salivary glands and the whole body, transcription initiation factor TFIID subunit was calculated as the most stably expressed gene by both programs. In addition, we compared the RNA-seq data with the results of geNorm and Normfinder and found that the stable genes revealed by RNA-seq analysis were also the ERGs recommended by geNorm and Normfinder. Furthermore, the use of the most stable gene suggested by RNA-seq analysis as an ERG produced similar gene expression patterns compared with results generated from the normalization against the most stable gene selected by geNorm and Normfinder and multiple genes recommended by geNorm. It indicates that RNA-seq data are reliable and provide a great source for ERG candidate exploration. Our results benefit future research on gene expression profiles of whiteflies and possibly other organisms.

  16. Applicability of the chymopapain gene used as endogenous reference gene for transgenic huanong no. 1 papaya detection.

    PubMed

    Guo, Jinchao; Yang, Litao; Liu, Xin; Zhang, Haibo; Qian, Bingjun; Zhang, Dabing

    2009-08-12

    The virus-resistant papaya (Carica papaya L.), Huanong no. 1, was the genetically modified (GM) fruit approved for growing in China in 2006. To implement the labeling regulation of GM papaya and its derivates, the development of papaya endogenous reference gene is very necessary for GM papaya detection. Herein, we reported one papaya specific gene, Chymopapain (CHY), as one suitable endogenous reference gene, used for GM papaya identification. Thereafter, we established the conventional and real-time quantitative PCR assays of the CHY gene. In the CHY conventional PCR assay, the limit of detection (LOD) was 25 copies of haploid papaya genome. In the CHY real-time quantitative PCR assay, both the LOD and the limit of quantification (LOQ) were as low as 12.5 copies of haploid papaya genome. Furthermore, we revealed the construct-specific sequence of Chinese GM papaya Huanong no. 1 and developed its conventional and quantitative PCR systems employing the CHY gene as endogenous reference gene. This work is useful for papaya specific identification and GM papaya detection.

  17. Metazoan mitochondrial gene sequence reference datasets for taxonomic assignment of environmental samples

    PubMed Central

    Machida, Ryuji J.; Leray, Matthieu; Ho, Shian-Lei; Knowlton, Nancy

    2017-01-01

    Mitochondrial-encoded genes are increasingly targeted in studies using high-throughput sequencing approaches for characterizing metazoan communities from environmental samples (e.g., plankton, meiofauna, filtered water). Yet, unlike nuclear ribosomal RNA markers, there is to date no high-quality reference dataset available for taxonomic assignments. Here, we retrieved all metazoan mitochondrial gene sequences from GenBank, and then quality filtered and formatted the datasets for taxonomic assignments using taxonomic assignment tools. The reference datasets—‘Midori references’—are available for download at www.reference-midori.info. Two versions are provided: (I) Midori-UNIQUE that contains all unique haplotypes associated with each species and (II) Midori-LONGEST that contains a single sequence, the longest, for each species. Overall, the mitochondrial Cytochrome oxidase subunit I gene was the most sequence-rich gene. However, sequences of the mitochondrial large ribosomal subunit RNA and Cytochrome b apoenzyme genes were observed for a large number of species in some phyla. The Midori reference is compatible with some taxonomic assignment software. Therefore, automated high-throughput sequence taxonomic assignments can be particularly effective using these datasets. PMID:28291235

  18. Recommended Reference Genes for Quantitative PCR Analysis in Soybean Have Variable Stabilities during Diverse Biotic Stresses.

    PubMed

    Bansal, Raman; Mittapelly, Priyanka; Cassone, Bryan J; Mamidala, Praveen; Redinbaugh, Margaret G; Michel, Andy

    2015-01-01

    For real-time reverse transcription-PCR (qRT-PCR) in soybean, reference genes in different tissues, developmental stages, various cultivars, and under stress conditions have been suggested but their usefulness for research on soybean under various biotic stresses occurring in North-Central U.S. is not known. Here, we investigated the expression stabilities of ten previously recommended reference genes (ABCT, CYP, EF1A, FBOX, GPDH, RPL30, TUA4, TUB4, TUA5, and UNK2) in soybean under biotic stress from Bean pod mottle virus (BPMV), powdery mildew (PMD), soybean aphid (SBA), and two-spotted spider mite (TSSM). BPMV, PMD, SBA, and TSSM are amongst the most common pest problems on soybean in North-Central U.S. and other regions. Reference gene stability was determined using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). Reference genes showed variability in their expression as well as stability across various stressors and the best reference genes were stress-dependent. ABCT and FBOX were found to be the most stable in soybean under both BPMV and SBA stress but these genes had only minimal to moderate stability during PMD and TSSM stress. Expression of TUA4 and CYP was found to be most stable during PMD stress; TUB4 and TUA4 were stable under TSSM stress. Under various biotic stresses on soybean analyzed, GPDH expression was found to be consistently unstable. For all biotic stressors on soybean, we obtained pairwise variation (V2/3) values less than 0.15 which suggested that combined use of the two most stable reference genes would be sufficient for normalization. Further, we demonstrated the utility of normalizing the qRT-PCR data for target genes using the most stable reference genes validated in current study. Following of the recommendations from our current study will enable an accurate and reliable normalization of qRT-PCR data in soybean under biotic stress.

  19. Recommended Reference Genes for Quantitative PCR Analysis in Soybean Have Variable Stabilities during Diverse Biotic Stresses

    PubMed Central

    Bansal, Raman; Mittapelly, Priyanka; Cassone, Bryan J.; Mamidala, Praveen; Redinbaugh, Margaret G.; Michel, Andy

    2015-01-01

    For real-time reverse transcription-PCR (qRT-PCR) in soybean, reference genes in different tissues, developmental stages, various cultivars, and under stress conditions have been suggested but their usefulness for research on soybean under various biotic stresses occurring in North-Central U.S. is not known. Here, we investigated the expression stabilities of ten previously recommended reference genes (ABCT, CYP, EF1A, FBOX, GPDH, RPL30, TUA4, TUB4, TUA5, and UNK2) in soybean under biotic stress from Bean pod mottle virus (BPMV), powdery mildew (PMD), soybean aphid (SBA), and two‐spotted spider mite (TSSM). BPMV, PMD, SBA, and TSSM are amongst the most common pest problems on soybean in North-Central U.S. and other regions. Reference gene stability was determined using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). Reference genes showed variability in their expression as well as stability across various stressors and the best reference genes were stress-dependent. ABCT and FBOX were found to be the most stable in soybean under both BPMV and SBA stress but these genes had only minimal to moderate stability during PMD and TSSM stress. Expression of TUA4 and CYP was found to be most stable during PMD stress; TUB4 and TUA4 were stable under TSSM stress. Under various biotic stresses on soybean analyzed, GPDH expression was found to be consistently unstable. For all biotic stressors on soybean, we obtained pairwise variation (V2/3) values less than 0.15 which suggested that combined use of the two most stable reference genes would be sufficient for normalization. Further, we demonstrated the utility of normalizing the qRT-PCR data for target genes using the most stable reference genes validated in current study. Following of the recommendations from our current study will enable an accurate and reliable normalization of qRT-PCR data in soybean under biotic stress. PMID:26244340

  20. Comprehensively identifying and characterizing the missing gene sequences in human reference genome with integrated analytic approaches.

    PubMed

    Chen, Geng; Wang, Charles; Shi, Leming; Tong, Weida; Qu, Xiongfei; Chen, Jiwei; Yang, Jianmin; Shi, Caiping; Chen, Long; Zhou, Peiying; Lu, Bingxin; Shi, Tieliu

    2013-08-01

    The human reference genome is still incomplete and a number of gene sequences are missing from it. The approaches to uncover them, the reasons causing their absence and their functions are less explored. Here, we comprehensively identified and characterized the missing genes of human reference genome with RNA-Seq data from 16 different human tissues. By using a combined approach of genome-guided transcriptome reconstruction coupled with genome-wide comparison, we uncovered 3.78 and 2.37 Mb transcribed regions in the human genome assemblies of Celera and HuRef either missed from their homologous chromosomes of NCBI human reference genome build 37.2 or partially or entirely absent from the reference. We further identified a significant number of novel transcript contigs in each tissue from de novo transcriptome assembly that are unalignable to NCBI build 37.2 but can be aligned to at least one of the genomes from Celera, HuRef, chimpanzee, macaca or mouse. Our analyses indicate that the missing genes could result from genome misassembly, transposition, copy number variation, translocation and other structural variations. Moreover, our results further suggest that a large portion of these missing genes are conserved between human and other mammals, implying their important biological functions. Totally, 1,233 functional protein domains were detected in these missing genes. Collectively, our study not only provides approaches for uncovering the missing genes of a genome, but also proposes the potential reasons causing genes missed from the genome and highlights the importance of uncovering the missing genes of incomplete genomes.

  1. Selection of reliable reference genes for quantitative real-time PCR gene expression analysis in Jute (Corchorus capsularis) under stress treatments.

    PubMed

    Niu, Xiaoping; Qi, Jianmin; Zhang, Gaoyang; Xu, Jiantang; Tao, Aifen; Fang, Pingping; Su, Jianguang

    2015-01-01

    To accurately measure gene expression using quantitative reverse transcription PCR (qRT-PCR), reliable reference gene(s) are required for data normalization. Corchorus capsularis, an annual herbaceous fiber crop with predominant biodegradability and renewability, has not been investigated for the stability of reference genes with qRT-PCR. In this study, 11 candidate reference genes were selected and their expression levels were assessed using qRT-PCR. To account for the influence of experimental approach and tissue type, 22 different jute samples were selected from abiotic and biotic stress conditions as well as three different tissue types. The stability of the candidate reference genes was evaluated using geNorm, NormFinder, and BestKeeper programs, and the comprehensive rankings of gene stability were generated by aggregate analysis. For the biotic stress and NaCl stress subsets, ACT7 and RAN were suitable as stable reference genes for gene expression normalization. For the PEG stress subset, UBC, and DnaJ were sufficient for accurate normalization. For the tissues subset, four reference genes TUBβ, UBI, EF1α, and RAN were sufficient for accurate normalization. The selected genes were further validated by comparing expression profiles of WRKY15 in various samples, and two stable reference genes were recommended for accurate normalization of qRT-PCR data. Our results provide researchers with appropriate reference genes for qRT-PCR in C. capsularis, and will facilitate gene expression study under these conditions.

  2. Selection of reliable reference genes for quantitative real-time PCR gene expression analysis in Jute (Corchorus capsularis) under stress treatments

    PubMed Central

    Niu, Xiaoping; Qi, Jianmin; Zhang, Gaoyang; Xu, Jiantang; Tao, Aifen; Fang, Pingping; Su, Jianguang

    2015-01-01

    To accurately measure gene expression using quantitative reverse transcription PCR (qRT-PCR), reliable reference gene(s) are required for data normalization. Corchorus capsularis, an annual herbaceous fiber crop with predominant biodegradability and renewability, has not been investigated for the stability of reference genes with qRT-PCR. In this study, 11 candidate reference genes were selected and their expression levels were assessed using qRT-PCR. To account for the influence of experimental approach and tissue type, 22 different jute samples were selected from abiotic and biotic stress conditions as well as three different tissue types. The stability of the candidate reference genes was evaluated using geNorm, NormFinder, and BestKeeper programs, and the comprehensive rankings of gene stability were generated by aggregate analysis. For the biotic stress and NaCl stress subsets, ACT7 and RAN were suitable as stable reference genes for gene expression normalization. For the PEG stress subset, UBC, and DnaJ were sufficient for accurate normalization. For the tissues subset, four reference genes TUBβ, UBI, EF1α, and RAN were sufficient for accurate normalization. The selected genes were further validated by comparing expression profiles of WRKY15 in various samples, and two stable reference genes were recommended for accurate normalization of qRT-PCR data. Our results provide researchers with appropriate reference genes for qRT-PCR in C. capsularis, and will facilitate gene expression study under these conditions. PMID:26528312

  3. Identification and validation of reference genes for quantitative real-time PCR studies in long yellow daylily, Hemerocallis citrina Borani

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gene expression analysis requires the use of reference genes in the target species. The long yellow daylily is rich in beneficial secondary metabolites and is considered as a functional vegetable. It is widely cultivated and consumed in East Asia. However, reference genes for use in RT-qPCR in this ...

  4. Evaluation of Reference Genes for Normalization of Gene Expression Using Quantitative RT-PCR under Aluminum, Cadmium, and Heat Stresses in Soybean.

    PubMed

    Gao, Mengmeng; Liu, Yaping; Ma, Xiao; Shuai, Qin; Gai, Junyi; Li, Yan

    2017-01-01

    Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used to analyze the relative gene expression level, however, the accuracy of qRT-PCR is greatly affected by the stability of reference genes, which is tissue- and environment- dependent. Therefore, choosing the most stable reference gene in a specific tissue and environment is critical to interpret gene expression patterns. Aluminum (Al), cadmium (Cd), and heat stresses are three important abiotic factors limiting soybean (Glycine max) production in southern China. To identify the suitable reference genes for normalizing the expression levels of target genes by qRT-PCR in soybean response to Al, Cd and heat stresses, we studied the expression stability of ten commonly used housekeeping genes in soybean roots and leaves under these three abiotic stresses, using five approaches, BestKeeper, Delta Ct, geNorm, NormFinder and RefFinder. We found TUA4 is the most stable reference gene in soybean root tips under Al stress. Under Cd stress, Fbox and UKN2 are the most stable reference genes in roots and leaves, respectively, while 60S is the most suitable reference gene when analyzing both roots and leaves together. For heat stress, TUA4 and UKN2 are the most stable housekeeping genes in roots and leaves, respectively, and UKN2 is the best reference gene for analysis of roots and leaves together. To validate the reference genes, we quantified the relative expression levels of six target genes that were involved in soybean response to Al, Cd or heat stresses, respectively. The expression patterns of these target genes differed between using the most and least stable reference genes, suggesting the selection of a suitable reference gene is critical for gene expression studies.

  5. Evaluation of Reference Genes for Normalization of Gene Expression Using Quantitative RT-PCR under Aluminum, Cadmium, and Heat Stresses in Soybean

    PubMed Central

    Ma, Xiao; Shuai, Qin; Gai, Junyi; Li, Yan

    2017-01-01

    Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used to analyze the relative gene expression level, however, the accuracy of qRT-PCR is greatly affected by the stability of reference genes, which is tissue- and environment- dependent. Therefore, choosing the most stable reference gene in a specific tissue and environment is critical to interpret gene expression patterns. Aluminum (Al), cadmium (Cd), and heat stresses are three important abiotic factors limiting soybean (Glycine max) production in southern China. To identify the suitable reference genes for normalizing the expression levels of target genes by qRT-PCR in soybean response to Al, Cd and heat stresses, we studied the expression stability of ten commonly used housekeeping genes in soybean roots and leaves under these three abiotic stresses, using five approaches, BestKeeper, Delta Ct, geNorm, NormFinder and RefFinder. We found TUA4 is the most stable reference gene in soybean root tips under Al stress. Under Cd stress, Fbox and UKN2 are the most stable reference genes in roots and leaves, respectively, while 60S is the most suitable reference gene when analyzing both roots and leaves together. For heat stress, TUA4 and UKN2 are the most stable housekeeping genes in roots and leaves, respectively, and UKN2 is the best reference gene for analysis of roots and leaves together. To validate the reference genes, we quantified the relative expression levels of six target genes that were involved in soybean response to Al, Cd or heat stresses, respectively. The expression patterns of these target genes differed between using the most and least stable reference genes, suggesting the selection of a suitable reference gene is critical for gene expression studies. PMID:28046130

  6. Selection of reliable reference genes for gene expression studies in Clonostachys rosea 67-1 under sclerotial induction.

    PubMed

    Sun, Zhan-Bin; Li, Shi-Dong; Sun, Man-Hong

    2015-07-01

    Reference genes are important to precisely quantify gene expression by real-time PCR. In order to identify stable and reliable expressed genes in mycoparasite Clonostachys rosea in different modes of nutrition, seven commonly used housekeeping genes, 18S rRNA, actin, β-tubulin, elongation factor 1, ubiquitin, ubiquitin-conjugating enzyme and glyceraldehyde-3-phosphate dehydrogenase, from the effective biocontrol isolate C. rosea 67-1 were tested for their expression under sclerotial induction and during vegetative growth on PDA medium. Analysis by three software programs showed that differences existed among the candidates. Elongation factor 1 was most stable; the M value in geNorm, SD value in Bestkeeper and stability value in Normfinder analysis were 0.405, 0.450 and 0.442, respectively, indicating that the gene elongation factor 1 could be used to normalize gene expression in C. rosea in both vegetative growth and parasitic process. By using elongation factor 1, the expression of a serine protease gene, sep, in different conditions was assessed, which was consistent with the transcriptomic data. This research provides an effective method to quantitate expression changes of target genes in C. rosea, and will assist in further investigation of parasitism-related genes of this fungus.

  7. Recommended reference genes for quantitative PCR analysis in soybean have variable stabilities during diverse biotic stresses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    For real-time reverse transcription-PCR (qRT-PCR) in soybean, reference genes in different tissues, developmental stages, various cultivars, and under stress conditions have been suggested but their usefulness for research on soybean under various biotic stresses occurring in North-Central U.S. is n...

  8. Evaluation of four genes in rice for their suitability as endogenous reference standards in quantitative PCR.

    PubMed

    Wang, Chong; Jiang, Lingxi; Rao, Jun; Liu, Yinan; Yang, Litao; Zhang, Dabing

    2010-11-24

    The genetically modified (GM) food/feed quantification depends on the reliable detection systems of endogenous reference genes. Currently, four endogenous reference genes including sucrose phosphate synthase (SPS), GOS9, phospholipase D (PLD), and ppi phosphofructokinase (ppi-PPF) of rice have been used in GM rice detection. To compare the applicability of these four rice reference genes in quantitative PCR systems, we analyzed the target nucleotide sequence variation in 58 conventional rice varieties from various geographic and phylogenic origins, also their quantification performances were evaluated using quantitative real-time PCR and GeNorm analysis via a series of statistical calculation to get a "M value" which is negative correlation with the stability of genes. The sequencing analysis results showed that the reported GOS9 and PLD taqman probe regions had detectable single nucleotide polymorphisms (SNPs) among the tested rice cultivars, while no SNPs were observed for SPS and ppi-PPF amplicons. Also, poor quantitative performance was detectable in these cultivars with SNPs using GOS9 and PLD quantitative PCR systems. Even though the PCR efficiency of ppi-PPF system was slightly lower, the SPS and ppi-PPF quantitative PCR systems were shown to be applicable for rice endogenous reference assay with less variation among the C(t) values, good reproducibility in quantitative assays, and the low M values by the comprehensive quantitative PCR comparison and GeNorm analysis.

  9. Selection of reference genes for expression analyses in liver of rats with impaired glucose metabolism

    PubMed Central

    Hernández, Alfonso H; Curi, Rui; Salazar, Luis A

    2015-01-01

    Hepatic gene expression studies are vital for identification of molecular factors involved in insulin resistance. However, the need of normalized gene expression data has led to the search of stable genes which are useful as a reference in specific experimental conditions. The aim of this study was to evaluate expression stability of potential reference genes for real-time PCR gene expression studies, in rats with insulin resistance, early programmed in intrauterine environment of maternal insulin resistance and triggered by exposure to a high sucrose and fat diet in adult life. Male rats coming from insulin resistant (F1IR) mothers or normal (F1N) mothers were fed a standard rodent diet from postnatal day 21 to day 56, and then divided in two groups each. One of each subgroups were fed a high sucrose and fat diet (groups F1IR + HSFD and F1N + HSFD respectively), the rest were fed a control diet (groups F1IR + CD and F1N + CD) for 14 days. Glucose metabolism related tests were later performed. After liver extraction, RNA was isolated and gene expression analyzes of seven potential reference genes (Actb, Gapdh, Gusb, Hprt1, Ldha, Rpl13a and Rplp1) were carried out. LinRegPCR software was used to analyze raw data and determinate baseline corrections, threshold lines, efficiency of PCR reactions and corrected Cq values. Evaluations of gene expression stabilities as well as the number of necessary genes for normalization were assessed with geNorm tool. All samples from all groups showed acceptable PCR amplification efficiencies. The most stable genes were Rplp1, Ldha, Hprt1 and Rpl13a and the less stable was Gapdh. For all groups, just 2 to 3 of the most stable genes were necessary for optimal gene expression data normalization in rat liver. Genes encoding ribosomal proteins are the most appropriated for normalization of expression data in the presented animal model. By contrast, Gapdh, one of the most used genes in normalization, is not recommendable due to its high

  10. Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis in Heliconius numata (Lepidoptera: Nymphalidae)

    PubMed Central

    Chouteau, Mathieu; Whibley, Annabel; Joron, Mathieu; Llaurens, Violaine

    2016-01-01

    Identifying the genetic basis of adaptive variation is challenging in non-model organisms and quantitative real time PCR. is a useful tool for validating predictions regarding the expression of candidate genes. However, comparing expression levels in different conditions requires rigorous experimental design and statistical analyses. Here, we focused on the neotropical passion-vine butterflies Heliconius, non-model species studied in evolutionary biology for their adaptive variation in wing color patterns involved in mimicry and in the signaling of their toxicity to predators. We aimed at selecting stable reference genes to be used for normalization of gene expression data in RT-qPCR analyses from developing wing discs according to the minimal guidelines described in Minimum Information for publication of Quantitative Real-Time PCR Experiments (MIQE). To design internal RT-qPCR controls, we studied the stability of expression of nine candidate reference genes (actin, annexin, eF1α, FK506BP, PolyABP, PolyUBQ, RpL3, RPS3A, and tubulin) at two developmental stages (prepupal and pupal) using three widely used programs (GeNorm, NormFinder and BestKeeper). Results showed that, despite differences in statistical methods, genes RpL3, eF1α, polyABP, and annexin were stably expressed in wing discs in late larval and pupal stages of Heliconius numata. This combination of genes may be used as a reference for a reliable study of differential expression in wings for instance for genes involved in important phenotypic variation, such as wing color pattern variation. Through this example, we provide general useful technical recommendations as well as relevant statistical strategies for evolutionary biologists aiming to identify candidate-genes involved adaptive variation in non-model organisms. PMID:27271971

  11. Identification of Reference Genes for Quantitative RT-PCR in Ascending Aortic Aneurysms

    PubMed Central

    Henn, Dominic; Bandner-Risch, Doris; Perttunen, Hilja; Schmied, Wolfram; Porras, Carlos; Ceballos, Francisco; Rodriguez-Losada, Noela; Schäfers, Hans-Joachim

    2013-01-01

    Hypertension and congenital aortic valve malformations are frequent causes of ascending aortic aneurysms. The molecular mechanisms of aneurysm formation under these circumstances are not well understood. Reference genes for gene activity studies in aortic tissue that are not influenced by aortic valve morphology and its hemodynamic consequences, aortic dilatation, hypertension, or antihypertensive medication are not available so far. This study determines genes in ascending aortic tissue that are independent of these parameters. Tissue specimens from dilated and undilated ascending aortas were obtained from 60 patients (age ≤70 years) with different morphologies of the aortic valve (tricuspid undilated n = 24, dilated n = 11; bicuspid undilated n = 6, dilated n = 15; unicuspid dilated n = 4). Of the studied individuals, 36 had hypertension, and 31 received ACE inhibitors or AT1 receptor antagonists. The specimens were obtained intraoperatively from the wall of the ascending aorta. We analyzed the expression levels of 32 candidate reference genes by quantitative RT-PCR (RT-qPCR). Differential expression levels were assessed by parametric statistics. The expression analysis of these 32 genes by RT-qPCR showed that EIF2B1, ELF1, and PPIA remained constant in their expression levels in the different specimen groups, thus being insensitive to aortic valve morphology, aortic dilatation, hypertension, and medication with ACE inhibitors or AT1 receptor antagonists. Unlike many other commonly used reference genes, the genes EIF2B1, ELF1, and PPIA are neither confounded by aortic comorbidities nor by antihypertensive medication and therefore are most suitable for gene expression analysis of ascending aortic tissue. PMID:23326585

  12. Construction of a standard reference plasmid containing seven target genes for the detection of transgenic cotton.

    PubMed

    Wang, Xujing; Tang, Qiaoling; Dong, Lei; Dong, Yufeng; Su, Yueyan; Jia, Shirong; Wang, Zhixing

    2014-07-01

    Insect resistance and herbicide tolerance are the dominant traits of commercialized transgenic cotton. In this study, we constructed a general standard reference plasmid for transgenic cotton detection. Target genes, including the cowpea trypsin gene cptI, the insect resistance gene cry1Ab/1Ac, the herbicide tolerance gene cp4-epsps, the Agrobacterium tumefaciens nopaline synthase (Nos) terminator that exists in transgenic cotton and part of the endogenous cotton SadI gene were amplified from plasmids pCPT1, pBT, pCP4 and pBI121 and from DNA of the nontransgenic cotton line K312, respectively. The genes cry1Ab/1Ac and cptI, as well as cp4-epsps and the Nos terminator gene, were ligated together to form the fusion genes cptI-Bt and cp4-Nos, respectively, by overlapping PCR. We checked the validity of genes Sad1, cptI-Bt and cp4-Nos by DNA sequencing. Then, positive clones of cptI-Bt, cp4-Nos and Sad1 were digested with the corresponding restriction enzymes and ligated sequentially into vector pCamBIA2300, which contains the CAMV 35S promoter and nptII gene, to form the reference plasmid pMCS. Qualitative detection showed that pMCS is a good positive control for transgenic cotton detection. Real-time PCR detection efficiencies with pMCS as a calibrator ranged from 94.35% to 98.67% for the standard curves of the target genes (R(2)⩾0.998). The relative standard deviation of the mean value for the known sample was 11.95%. These results indicate that the strategy of using the pMCS plasmid as a reference material is feasible and reliable for the detection of transgenic cotton. Therefore, this plasmid can serve as a useful reference tool for qualitative and quantitative detection of single or stacked trait transgenic cotton, thus paving the way for the identification of various products containing components of transgenic cotton.

  13. Identification and Evaluation of Reference Genes for Accurate Transcription Normalization in Safflower under Different Experimental Conditions

    PubMed Central

    Li, Dandan; Hu, Bo; Wang, Qing; Liu, Hongchang; Pan, Feng; Wu, Wei

    2015-01-01

    Safflower (Carthamus tinctorius L.) has received a significant amount of attention as a medicinal plant and oilseed crop. Gene expression studies provide a theoretical molecular biology foundation for improving new traits and developing new cultivars. Real-time quantitative PCR (RT-qPCR) has become a crucial approach for gene expression analysis. In addition, appropriate reference genes (RGs) are essential for accurate and rapid relative quantification analysis of gene expression. In this study, fifteen candidate RGs involved in multiple metabolic pathways of plants were finally selected and validated under different experimental treatments, at different seed development stages and in different cultivars and tissues for real-time PCR experiments. These genes were ABCS, 60SRPL10, RANBP1, UBCL, MFC, UBCE2, EIF5A, COA, EF1-β, EF1, GAPDH, ATPS, MBF1, GTPB and GST. The suitability evaluation was executed by the geNorm and NormFinder programs. Overall, EF1, UBCE2, EIF5A, ATPS and 60SRPL10 were the most stable genes, and MBF1, as well as MFC, were the most unstable genes by geNorm and NormFinder software in all experimental samples. To verify the validation of RGs selected by the two programs, the expression analysis of 7 CtFAD2 genes in safflower seeds at different developmental stages under cold stress was executed using different RGs in RT-qPCR experiments for normalization. The results showed similar expression patterns when the most stable RGs selected by geNorm or NormFinder software were used. However, the differences were detected using the most unstable reference genes. The most stable combination of genes selected in this study will help to achieve more accurate and reliable results in a wide variety of samples in safflower. PMID:26457898

  14. Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stresses

    PubMed Central

    Zhang, Yunxing; Han, Xiaojiao; Chen, Shuangshuang; Zheng, Liu; He, Xuelian; Liu, Mingying; Qiao, Guirong; Wang, Yang; Zhuo, Renying

    2017-01-01

    Salix matsudana is a deciduous, rapidly growing willow species commonly cultivated in China, which can tolerate drought, salt, and heavy metal stress conditions. Selection of suitable reference genes for quantitative real-time PCR is important for normalizing the expression of the key genes associated with various stresses. To validate suitable reference genes, we selected 11 candidate reference genes (five traditional housekeeping genes and six novel genes) and analyzed their expression stability in various samples, including different tissues and under different abiotic stress treatments. The expression of these genes was determined using five programs—geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder. The results showed that α-TUB2 (alpha-tubulin 2) and DnaJ (chaperone protein DnaJ 49) were the most stable reference genes across all the tested samples. We measured the expression profiles of the defense response gene SmCAT (catalase) using the two most stable and one least stable reference genes in all samples of S. matsudana. The relative quantification of SmCAT varied greatly according to the different reference genes. We propose that α-TUB2 and DnaJ should be the preferred reference genes for normalization and quantification of transcript levels in future gene expression studies in willow species under various abiotic stress conditions. PMID:28120870

  15. Reference Genes in the Pathosystem Phakopsora pachyrhizi/ Soybean Suitable for Normalization in Transcript Profiling

    PubMed Central

    Hirschburger, Daniela; Müller, Manuel; Voegele, Ralf T.; Link, Tobias

    2015-01-01

    Phakopsora pachyrhizi is a devastating pathogen on soybean, endangering soybean production worldwide. Use of Host Induced Gene Silencing (HIGS) and the study of effector proteins could provide novel strategies for pathogen control. For both approaches quantification of transcript abundance by RT-qPCR is essential. Suitable stable reference genes for normalization are indispensable to obtain accurate RT-qPCR results. According to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and using algorithms geNorm and NormFinder we tested candidate reference genes from P. pachyrhizi and Glycine max for their suitability in normalization of transcript levels throughout the infection process. For P. pachyrhizi we recommend a combination of CytB and PDK or GAPDH for in planta experiments. Gene expression during in vitro stages and over the whole infection process was found to be highly unstable. Here, RPS14 and UbcE2 are ranked best by geNorm and NormFinder. Alternatively CytB that has the smallest Cq range (Cq: quantification cycle) could be used. We recommend specification of gene expression relative to the germ tube stage rather than to the resting urediospore stage. For studies omitting the resting spore and the appressorium stages a combination of Elf3 and RPS9, or PKD and GAPDH should be used. For normalization of soybean genes during rust infection Ukn2 and cons7 are recommended. PMID:26404265

  16. Genome-wide identification and characterization of reference genes with different transcript abundances for Streptomyces coelicolor

    PubMed Central

    Li, Shanshan; Wang, Weishan; Li, Xiao; Fan, Keqiang; Yang, Keqian

    2015-01-01

    The lack of reliable reference genes (RGs) in the genus Streptomyces hampers effort to obtain the precise data of transcript levels. To address this issue, we aimed to identify reliable RGs in the model organism Streptomyces coelicolor. A pool of potential RGs containing 1,471 genes was first identified by determining the intersection of genes with stable transcript levels from four time-series transcriptome microarray datasets of S. coelicolor M145 cultivated in different conditions. Then, following a strict rational selection scheme including homology analysis, disturbance analysis, function analysis and transcript abundance analysis, 13 candidates were selected from the 1,471 genes. Based on real-time quantitative reverse transcription PCR assays, SCO0710, SCO6185, SCO1544, SCO3183 and SCO4758 were identified as the top five genes with the most stable transcript levels among the 13 candidates. Further analyses showed these five genes also maintained stable transcript levels in different S. coelicolor strains, as well as in Streptomyces avermitilis MA-4680 and Streptomyces clavuligerus NRRL 3585, suggesting they could fulfill the requirements of accurate data normalization in streptomycetes. Moreover, the systematic strategy employed in this work could be used for reference in other microorganism to select reliable RGs. PMID:26527303

  17. [Selection of reference genes for quantitative real-time PCR in six oil-tea camellia based on RNA].

    PubMed

    Zhou, C F; Lin, P; Yao, X H; Wang, K L; Chang, J; Han, X J

    2013-01-01

    qRT-PCR is becoming a routine tool in molecular biology to study gene expression. It is nec- essary to find stable reference genes when performing qRT-PCR. The expression of genes cloned in oil-tea camellia currently can't be accurately analyzed because of a lack of suitable reference genes. We collected different tissues (including roots, stems, leaves, flowers and seeds) from six oil-tea camellia species to determine stable reference genes. Five novel and ten traditional reference gene sequences were selected from the RNA-seq database of Camellia oleifera C. Abel seeds and specific PCR primers were designed for each. Cycle threshold (Ct) data were obtained from each reaction for all samples. Three different software tools, geNorm, NormFinder and BestKeeper were applied to calculate the expression stability of the candidate reference genes according to the Ct values. The results were similar between analyzed by the three software packages, and indicated that the traditional gene TUBa-3, AC17a and the novel gene CESA were relatively stable in all species and tissues. However, no genes were sufficiently stable across all species and tissues, thus the optimal number of reference genes required for accurate normalization varied from two to six. Finally, the relative expression ofsqualene synthase (SQS) and squalene epoxidase (SQE) genes related to important ingredients squalene and tea saponin in oil-tea camellia seeds were compared by using stable to less stable reference genes. The comparison results validated the selection of reference genes in the current study. In summary, different optimal numbers of suitable reference genes were found for the different tissues of six oil-tea camellia species.

  18. Selection of Reference Genes for Quantitative Real-Time PCR during Flower Development in Tree Peony (Paeonia suffruticosa Andr.)

    PubMed Central

    Li, Jian; Han, Jigang; Hu, Yonghong; Yang, Ji

    2016-01-01

    Tree peony (Paeonia suffruticosa) is a perennial plant indigenous to China known for its elegant and vibrantly colorful flowers. A few genes involved in petal pigmentation have been cloned in tree peony. However, to date, there have been few studies on the comparison and selection of stable reference genes for gene expression analysis by quantitative reverse-transcription PCR (qRT-PCR) in this species. In this study, 10 candidate reference genes were evaluated for the normalization of qRT-PCR in three tree peony cultivars. GAPDH and UBC were identified as the top two most stable reference genes in ‘Feng Dan’ and ‘Xi Shi,’ and EF-1α/UBC was recommended to be the best combination for ‘Que Hao.’ The expression stability of various reference genes differed across cultivars, suggesting that selection and validation of reliable reference genes for quantitative gene expression analysis was necessary not only for different species but also for different cultivars. The results provided a list of reference genes for further study on gene expression in P. suffruticosa. However, in any case, a preliminary check on the accuracy of the best performing reference genes is requested for each qRT-PCR experiment. PMID:27148337

  19. Identification and validation of reference genes for quantitative real-time polymerase chain reaction in Cimex lectularius.

    PubMed

    Mamidala, Praveen; Rajarapu, Swapna P; Jones, Susan C; Mittapalli, Omprakash

    2011-07-01

    Quantitative real-time polymerase chain reaction (qRT-PCR) has emerged as robust methodology for gene expression studies, but reference genes are crucial for accurate normalization. Commonly used reference genes are housekeeping genes that are thought to be nonregulated; however, their expression can be unstable across different experimental conditions. We report the identification and validation of suitable reference genes in the bed bug, Cimex lectularius, by using qRT-PCR. The expression stability of eight reference genes in different tissues (abdominal cuticle, midgut, Malpighian tubules, and ovary) and developmental stages (early instar nymphs, late instar nymphs, and adults) of pesticide-susceptible and pesticide-exposed C. lectularius were analyzed using geNorm, NormFinder, and BestKeeper. Overall expression analysis of the eight reference genes revealed significant variation among samples, indicating the necessity of validating suitable reference genes for accurate quantification of mRNA transcripts. Ribosomal protein (RPL18) exhibited the most stable gene expression across all the tissue and developmental-stage samples; a-tubulin revealed the least stability across all of the samples examined. Thus, we recommend RPL18 as a suitable reference gene for normalization in gene expression studies of C. lectularius.

  20. Identification and Validation of Reference Genes for Quantitative Real-Time PCR Normalization and Its Applications in Lycium

    PubMed Central

    Zeng, Shaohua; Liu, Yongliang; Wu, Min; Liu, Xiaomin; Shen, Xiaofei; Liu, Chunzhao; Wang, Ying

    2014-01-01

    Lycium barbarum and L. ruthenicum are extensively used as traditional Chinese medicinal plants. Next generation sequencing technology provides a powerful tool for analyzing transcriptomic profiles of gene expression in non-model species. Such gene expression can then be confirmed with quantitative real-time polymerase chain reaction (qRT-PCR). Therefore, use of systematically identified suitable reference genes is a prerequisite for obtaining reliable gene expression data. Here, we calculated the expression stability of 18 candidate reference genes across samples from different tissues and grown under salt stress using geNorm and NormFinder procedures. The geNorm-determined rank of reference genes was similar to those defined by NormFinder with some differences. Both procedures confirmed that the single most stable reference gene was ACNTIN1 for L. barbarum fruits, H2B1 for L. barbarum roots, and EF1α for L. ruthenicum fruits. PGK3, H2B2, and PGK3 were identified as the best stable reference genes for salt-treated L. ruthenicum leaves, roots, and stems, respectively. H2B1 and GAPDH1+PGK1 for L. ruthenicum and SAMDC2+H2B1 for L. barbarum were the best single and/or combined reference genes across all samples. Finally, expression of salt-responsive gene NAC, fruit ripening candidate gene LrPG, and anthocyanin genes were investigated to confirm the validity of the selected reference genes. Suitable reference genes identified in this study provide a foundation for accurately assessing gene expression and further better understanding of novel gene function to elucidate molecular mechanisms behind particular biological/physiological processes in Lycium. PMID:24810586

  1. DEVELOPMENT OF CRASSPHAGE-BASED QPCR ASSAYS ...

    EPA Pesticide Factsheets

    A newly discovered bacteriophage, “crAssphage”, is predicted to be both highlyabundant and predominantly human-associated, both ideal characteristics for a human-specific fecal indicator. A total of 384 end-point PCR primers were designed along the length of the crAssphage genome, eliminating regions suspected to be hypervariable or react with other animal sources. The primer pairs were rigorously tested in three rounds of screening for specificity, geographic variability, limit of detection, and environmental water performance. The two best performing assays, crAss056 and crAss064, were adapted to a qPCR platform and exhibited a specificity of 98.0% and 98.9%, respectively. The markers’ abundance was compared with two bacterial based assays and were found at concentrations at or above the bacterial based assays in wastewater influent and impacted environmental waters. This poster will present the methodology of the novel marker development and the potential uses for this technology in maintaining sustainable waterways in the future. To inform the public.

  2. LbCML38 and LbRH52, two reference genes derived from RNA-Seq data suitable for assessing gene expression in Lycium barbarum L.

    PubMed Central

    Gong, Lei; Yang, Yajun; Chen, Yuchao; Shi, Jing; Song, Yuxia; Zhang, Hongxia

    2016-01-01

    For quantitative real-time PCR (qRT-PCR) analysis, the key prerequisite that determines result accuracy is the selection of appropriate reference gene(s). Goji (Lycium barbarum L.) is a multi-branched shrub belonging to the Solanaceae family. To date, no systematic screening or evaluation of reference gene(s) in Goji has been performed. In this work, we identified 18 candidate reference genes from the transcriptomic sequencing data of 14 samples of Goji at different developmental stages and under drought stress condition. The expression stability of these candidate genes was rigorously analyzed using qRT-PCR and four different statistical algorithms: geNorm, BestKeeper, NormFinder and RefFinder. Two novel reference genes LbCML38 and LbRH52 showed the most stable expression, whereas the traditionally used reference genes such as LbGAPDH, LbHSP90 and LbTUB showed unstable expression in the tested samples. Expression of a target gene LbMYB1 was also tested and compared using optimal reference genes LbCML38 and LbRH52, mediocre reference gene LbActin7, and poor reference gene LbHSP90 as normalization standards, respectively. As expected, calculation of the target gene expression by normalization against LbCML38, LbActin7 or LbHSP90 showed significant differences. Our findings suggest that LbCML38 and LbRH52 can be used as reference genes for gene expression analysis in Goji. PMID:27841319

  3. Evaluation of reference genes in Vibrio parahaemolyticus for gene expression analysis using quantitative RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize the viru...

  4. Comprehensive evaluation of candidate reference genes for qRT-PCR studies of gene expression in mustard aphid, Lipaphis erysimi (Kalt)

    PubMed Central

    Koramutla, Murali Krishna; Aminedi, Raghavendra; Bhattacharya, Ramcharan

    2016-01-01

    Mustard aphid, also known as turnip aphid (Lipaphis erysimi) is a major insect pest of rapeseed-mustard group of crops. Tremendous economic significance has led to substantial basic research involving gene-expression studies in this insect species. In qRT-PCR analysis of gene-expression, normalization of data against RNA variation by using appropriate reference gene is fundamental. However, appropriate reference genes are not known in case of L. erysimi. We evaluated 11 candidate reference genes for their expression stability in 21 samples of L. erysimi subjected to various regimes of experimental treatments. Unlike other studies, we validated true effects of the treatments on the samples either by gene-expression study of an associated marker gene or by biochemical tests. In the validated samples, expression stability of the reference genes was analysed by employing four different statistical softwares geNorm, NormFinder, BestKeeper and deltaCt. Drawing consensus on the results from different softwares, we recommend three best reference genes 16S, RPS18 and RPL13 for normalization of qRT-PCR data in L. erysimi. This study provides for the first time a comprehensive list of suitable reference genes for mustard aphid and demonstrates the advantage of using more than one reference gene in combination for certain experimental conditions. PMID:27165720

  5. Sources of Blood Meals of Sylvatic Triatoma guasayana near Zurima, Bolivia, Assayed with qPCR and 12S Cloning

    PubMed Central

    Lucero, David E.; Ribera, Wilma; Pizarro, Juan Carlos; Plaza, Carlos; Gordon, Levi W.; Peña, Reynaldo; Morrissey, Leslie A.; Rizzo, Donna M.; Stevens, Lori

    2014-01-01

    Background In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia. Methodology/Principal Findings We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors). Conclusions/Significance We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors. PMID:25474154

  6. A gene-based association method for mapping traits using reference transcriptome data

    PubMed Central

    Gamazon, Eric R.; Wheeler, Heather E.; Shah, Kaanan P.; Mozaffari, Sahar V.; Aquino-Michaels, Keston; Carroll, Robert J.; Eyler, Anne E.; Denny, Joshua C.; Nicolae, Dan L.; Cox, Nancy J.; Kyung Im, Hae

    2015-01-01

    Genome-wide association studies (GWAS) have identified thousands of variants robustly associated with complex traits. However, the biological mechanisms underlying these associations are, in general, not well understood. We propose a gene-based association method called PrediXcan that directly tests the molecular mechanisms through which genetic variation affects phenotype. The approach estimates the component of gene expression determined by an individual’s genetic profile and correlates the “imputed” gene expression with the phenotype under investigation to identify genes involved in the etiology of the phenotype. The genetically regulated gene expression is estimated using whole-genome tissue-dependent prediction models trained with reference transcriptome datasets. PrediXcan enjoys the benefits of gene-based approaches such as reduced multiple testing burden and a principled approach to the design of follow-up experiments. Our results demonstrate that PrediXcan can detect known and novel genes associated with disease traits and provide insights into the mechanism of these associations. PMID:26258848

  7. Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Analysis of Gene Expression in Lycoris aurea

    PubMed Central

    Ma, Rui; Xu, Sheng; Zhao, Yucheng; Xia, Bing; Wang, Ren

    2016-01-01

    Lycoris aurea (L' Hér.) Herb, a perennial grass species, produces a unique variety of pharmacologically active Amaryllidaceae alkaloids. However, the key enzymes and their expression pattern involved in the biosynthesis of Amaryllidaceae alkaloids (especially for galanthamine) are far from being fully understood. Quantitative real-time polymerase chain reaction (qRT-PCR), a commonly used method for quantifying gene expression, requires stable reference genes to normalize its data. In this study, to choose the appropriate reference genes under different experimental conditions, 14 genes including YLS8 (mitosis protein YLS8), CYP2 (Cyclophilin 2), CYP 1 (Cyclophilin 1), TIP41 (TIP41-like protein), EXP2 (Expressed protein 2), PTBP1 (Polypyrimidine tract-binding protein 1), EXP1 (Expressed protein 1), PP2A (Serine/threonine-protein phosphatase 2A), β-TUB (β-tubulin), α-TUB (α-tubulin), EF1-α (Elongation factor 1-α), UBC (Ubiquitin-conjugating enzyme), ACT (Actin) and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were selected from the transcriptome datasets of L. aurea. And then, expressions of these genes were assessed by qRT-PCR in various tissues and the roots under different treatments. The expression stability of the 14 candidates was analyzed by three commonly used software programs (geNorm, NormFinder, and BestKeeper), and their results were further integrated into a comprehensive ranking based on the geometric mean. The results show the relatively stable genes for each subset as follows: (1) EXP1 and TIP41 for all samples; (2) UBC and EXP1 for NaCl stress; (3) PTBP1 and EXP1 for heat stress, polyethylene glycol (PEG) stress and ABA treatment; (4) UBC and CYP2 for cold stress; (5) PTBP1 and PP2A for sodium nitroprusside (SNP) treatment; (6) CYP1 and TIP41 for methyl jasmonate (MeJA) treatment; and (7) EXP1 and TIP41 for various tissues. The reliability of these results was further enhanced through comparison between part qRT-PCR result and RNA

  8. Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae)

    PubMed Central

    2010-01-01

    Background Quantitative real-time reverse transcriptase PCR (RT-qPCR) has been widely used for quantification of mRNA as a way to determine key genes involved in different biological processes. For accurate gene quantification analysis, normalization of RT-qPCR data is absolutely essential. To date, normalization is most frequently achieved by the use of internal controls, often referred to as reference genes. However, several studies have shown that the reference genes used for the quantification of mRNA expression can be affected by the experimental set-up or cell type resulting in variation of the expression level of these key genes. Therefore, the evaluation of reference genes is critical for gene expression profiling, which is often neglected in gene expression studies of insects. For this purpose, ten candidate reference genes were investigated in three different tissues (midgut, Malpighian tubules, and fat body) of the oriental fruit fly, Bactrocera dorsalis (Hendel). Results Two different programs, geNorm and Normfinder, were used to analyze the data. According to geNorm, α-TUB + ACT5 are the most appropriate reference genes for gene expression profiling across the three different tissues in the female flies, while ACT3 + α-TUB are considered as the best for males. Furthermore, we evaluated the stability of the candidate reference genes to determine the sexual differences in the same tissue. In the midgut and Malpighian tubules, ACT2 + α-TUB are the best choice for both males and females. However, α-TUB + ACT1 are the best pair for fat body. Meanwhile, the results calculated by Normfinder are quite the same as the results with geNorm; α-TUB is always one of the most stable genes in each sample validated by the two programs. Conclusions In this study, we validated the suitable reference genes for gene expression profiling in different tissues of B. dorsalis. Moreover, appropriate reference genes were selected out for gene expression profiling of the

  9. SYBR®Green qPCR Salmonella detection system allowing discrimination at the genus, species and subspecies levels.

    PubMed

    Barbau-Piednoir, Elodie; Bertrand, Sophie; Mahillon, Jacques; Roosens, Nancy H; Botteldoorn, Nadine

    2013-11-01

    In this work, a three-level Salmonella detection system based on a combination of seven SYBR®Green qPCR was developed. This detection system discriminates Salmonella at the genus, species and subspecies levels using a single 96-well plate. The SYBR®Green qPCR assays target the invA, rpoD, iroB and safC genes, as well as the STM0296 locus, putatively coding for a cytoplasmic protein. This study includes the design of primer pairs, in silico and in situ selectivity, sensitivity, repeatability and reproducibility evaluations of the seven SYBR®Green qPCR assays. Each detection level displayed a selectivity of 100 %. This combinatory SYBR®Green qPCR system was also compared with three commercially available Salmonella qPCR detection kits. This comparison highlighted the importance of using a multi-gene detection system to be able to detect every target strain, even those with deletion or mutation of important genes.

  10. Reference materials (RMs) for analysis of the human factor II (prothrombin) gene G20210A mutation.

    PubMed

    Klein, Christoph L; Márki-Zay, János; Corbisier, Philippe; Gancberg, David; Cooper, Susan; Gemmati, Donato; Halbmayer, Walter-Michael; Kitchen, Steve; Melegh, Béla; Neumaier, Michael; Oldenburg, Johannes; Leibundgut, Elisabeth Oppliger; Reitsma, Pieter H; Rieger, Sandra; Schimmel, Heinz G; Spannagl, Michael; Tordai, Attilia; Tosetto, Alberto; Visvikis, Sophie; Zadro, Renata; Mannhalter, Christine

    2005-01-01

    The Scientific Committee of Molecular Biology Techniques (C-MBT) in Clinical Chemistry of the IFCC has initiated a joint project in co-operation with the European Commission, Joint Research Centre, Institute of Reference Materials and Measurements to develop and produce plasmid-type reference materials (RMs) for the analysis of the human prothrombin gene G20210A mutation. Although DNA tests have a high impact on clinical decision-making and the number of tests performed in diagnostic laboratories is high, issues of quality and quality assurance exist, and currently only a few RMs for clinical genetic testing are available. A gene fragment chosen was produced that spans all primer annealing sites published to date. Both the wild-type and mutant alleles of this gene fragment were cloned into a pUC18 plasmid and two plasmid RMs were produced. In addition, a mixture of both plasmids was produced to mimic the heterozygous genotype. The present study describes the performance of these reference materials in a commutability study, in which they were tested by nine different methods in 13 expert laboratories. This series of plasmid RMs are, to the best of our knowledge, the first plasmid-type clinical genetic RMs introduced worldwide.

  11. Identification and Validation of Reference Genes for RT-qPCR Analysis in Non-Heading Chinese Cabbage Flowers

    PubMed Central

    Wang, Cheng; Cui, Hong-Mi; Huang, Tian-Hong; Liu, Tong-Kun; Hou, Xi-Lin; Li, Ying

    2016-01-01

    Non-heading Chinese cabbage (Brassica rapa ssp. chinensis Makino) is an important vegetable member of Brassica rapa crops. It exhibits a typical sporophytic self-incompatibility (SI) system and is an ideal model plant to explore the mechanism of SI. Gene expression research are frequently used to unravel the complex genetic mechanism and in such studies appropriate reference selection is vital. Validation of reference genes have neither been conducted in Brassica rapa flowers nor in SI trait. In this study, 13 candidate reference genes were selected and examined systematically in 96 non-heading Chinese cabbage flower samples that represent four strategic groups in compatible and self-incompatible lines of non-heading Chinese cabbage. Two RT-qPCR analysis software, geNorm and NormFinder, were used to evaluate the expression stability of these genes systematically. Results revealed that best-ranked references genes should be selected according to specific sample subsets. DNAJ, UKN1, and PP2A were identified as the most stable reference genes among all samples. Moreover, our research further revealed that the widely used reference genes, CYP and ACP, were the least suitable reference genes in most non-heading Chinese cabbage flower sample sets. To further validate the suitability of the reference genes identified in this study, the expression level of SRK and Exo70A1 genes which play important roles in regulating interaction between pollen and stigma were studied. Our study presented the first systematic study of reference gene(s) selection for SI study and provided guidelines to obtain more accurate RT-qPCR results in non-heading Chinese cabbage. PMID:27375663

  12. Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs.

    PubMed

    Park, Sang-Je; Kwon, Seul Gi; Hwang, Jung Hye; Park, Da Hye; Kim, Tae Wan; Kim, Chul Wook

    2015-03-01

    Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most reliable molecular biology technique for assessment of mRNA expression levels. However, to obtain the accurate RT-qPCR results, the expression levels of genes of interest should be normalized with appropriate reference genes and optimal numbers of reference genes. In this study, we assessed the expression stability of 15 well-known candidate reference genes (ACTB, ALDOA, B2M, GAPDH, HPAR1, HSPCB, PGK1, POLR2G, PPIA, RPL4, RPS18, SDHA, TBP, TOP2B, and YWHAZ) in seven body tissues (liver, lung, kidney, spleen, stomach, small intestine, and large intestine) of Berkshire, Landrace, Duroc, and Yorkshire pigs using three excel-based programs, geNorm, NormFinder, and BestKeeper. Combination analysis of these three programs showed that the stable and appropriate reference genes are PPIA, TBP, and HSPCB in Berkshire pigs; PPIA, TBP, RPL4, and RPS18 in Landrace pigs; PPIA and TBP in Duroc pigs; and PPIA, TOP2B, RPL4, and RPS18 in Yorkshire pigs. Because the four pig breeds had different suitable reference genes, the selection of appropriate reference genes is essential in RT-qPCR analyses. Taken together, our data could help to select reliable reference genes for the normalization of expression levels of various target genes in pigs.

  13. Low-level lasers and mRNA levels of reference genes used in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Teixeira, A. F.; Machado, Y. L. R. C.; Fonseca, A. S.; Mencalha, A. L.

    2016-11-01

    Low-level lasers are widely used for the treatment of diseases and antimicrobial photodynamic therapy. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is widely used to evaluate mRNA levels and output data from a target gene are commonly relative to a reference mRNA that cannot vary according to treatment. In this study, the level of reference genes from Escherichia coli exposed to red or infrared lasers at different fluences was evaluated. E. coli AB1157 cultures were exposed to red (660 nm) and infrared (808 nm) lasers, incubated (20 min, 37 °C), the total RNA was extracted, and cDNA synthesis was performed to evaluate mRNA levels from arcA, gyrA and rpoA genes by RT-qPCR. Melting curves and agarose gel electrophoresis were carried out to evaluate specific amplification. Data were analyzed by geNorm, NormFinder and BestKeeper. The melting curve and agarose gel electrophoresis showed specific amplification. Although mRNA levels from arcA, gyrA or rpoA genes presented no significant variations trough a traditional statistical analysis, Excel-based tools revealed that these reference genes are not suitable for E. coli cultures exposed to lasers. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from arcA, gyrA and rpoA in E. coli cells.

  14. Validation of qPCR Methods for the Detection of Mycobacterium in New World Animal Reservoirs.

    PubMed

    Housman, Genevieve; Malukiewicz, Joanna; Boere, Vanner; Grativol, Adriana D; Pereira, Luiz Cezar M; Silva, Ita de Oliveira; Ruiz-Miranda, Carlos R; Truman, Richard; Stone, Anne C

    2015-11-01

    Zoonotic pathogens that cause leprosy (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis complex, MTBC) continue to impact modern human populations. Therefore, methods able to survey mycobacterial infection in potential animal hosts are necessary for proper evaluation of human exposure threats. Here we tested for mycobacterial-specific single- and multi-copy loci using qPCR. In a trial study in which armadillos were artificially infected with M. leprae, these techniques were specific and sensitive to pathogen detection, while more traditional ELISAs were only specific. These assays were then employed in a case study to detect M. leprae as well as MTBC in wild marmosets. All marmosets were negative for M. leprae DNA, but 14 were positive for the mycobacterial rpoB gene assay. Targeted capture and sequencing of rpoB and other MTBC genes validated the presence of mycobacterial DNA in these samples and revealed that qPCR is useful for identifying mycobacterial-infected animal hosts.

  15. Identification and validation of reference genes for expression studies in human keratinocyte cell lines treated with and without interferon-γ - a method for qRT-PCR reference gene determination.

    PubMed

    Riemer, Angelika B; Keskin, Derin B; Reinherz, Ellis L

    2012-08-01

    Based on the exquisite sensitivity, reproducibility and wide dynamic range of quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR), it is currently the gold standard for gene expression studies. Target gene expression is calculated relative to a stably expressed reference gene. An ideal reference should be uniformly expressed during all experimental conditions within the given experimental system. However, no commonly applicable 'best' reference gene has been identified. Thus, endogenous controls must be determined for every experimental system. As no appropriate reference genes have been reported for immunological studies in keratinocytes, we aimed at identifying and validating a set of endogenous controls for these settings. An extensive validation of sixteen possible endogenous controls in a panel of 8 normal and transformed keratinocyte cell lines in experimental conditions with and without interferon-γ was performed. RNA and cDNA quality was stringently controlled. Candidate reference genes were assessed by TaqMan(®) qRT-PCR. Two different statistical algorithms were used to determine the most stably and reproducibly expressed housekeeping genes. mRNA abundance was compared and reference genes with widely different ranges of expression than possible target genes were excluded. Subsequent geNorm and NormFinder analyses identified GAPDH, PGK1, IPO8 and PPIA as the most stably expressed genes in the keratinocyte panel under the given experimental conditions. We conclude that the geometric means of expression values of these four genes represents a robust normalization factor for qRT-PCR analyses in interferon-γ-dependent gene expression studies in keratinocytes. The methodology and results herein may help other researchers by facilitating their choice of reference genes.

  16. Reference gene selection for quantitative real-time PCR in Solanum lycopersicum L. inoculated with the mycorrhizal fungus Rhizophagus irregularis.

    PubMed

    Fuentes, Alejandra; Ortiz, Javier; Saavedra, Nicolás; Salazar, Luis A; Meneses, Claudio; Arriagada, Cesar

    2016-04-01

    The gene expression stability of candidate reference genes in the roots and leaves of Solanum lycopersicum inoculated with arbuscular mycorrhizal fungi was investigated. Eight candidate reference genes including elongation factor 1 α (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), protein phosphatase 2A (PP2Acs), ribosomal protein L2 (RPL2), β-tubulin (TUB), ubiquitin (UBI) and actin (ACT) were selected, and their expression stability was assessed to determine the most stable internal reference for quantitative PCR normalization in S. lycopersicum inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. The stability of each gene was analysed in leaves and roots together and separated using the geNorm and NormFinder algorithms. Differences were detected between leaves and roots, varying among the best-ranked genes depending on the algorithm used and the tissue analysed. PGK, TUB and EF1 genes showed higher stability in roots, while EF1 and UBI had higher stability in leaves. Statistical algorithms indicated that the GAPDH gene was the least stable under the experimental conditions assayed. Then, we analysed the expression levels of the LePT4 gene, a phosphate transporter whose expression is induced by fungal colonization in host plant roots. No differences were observed when the most stable genes were used as reference genes. However, when GAPDH was used as the reference gene, we observed an overestimation of LePT4 expression. In summary, our results revealed that candidate reference genes present variable stability in S. lycopersicum arbuscular mycorrhizal symbiosis depending on the algorithm and tissue analysed. Thus, reference gene selection is an important issue for obtaining reliable results in gene expression quantification.

  17. Deep RNA sequencing analysis of readthrough gene fusions in human prostate adenocarcinoma and reference samples

    PubMed Central

    2011-01-01

    Background Readthrough fusions across adjacent genes in the genome, or transcription-induced chimeras (TICs), have been estimated using expressed sequence tag (EST) libraries to involve 4-6% of all genes. Deep transcriptional sequencing (RNA-Seq) now makes it possible to study the occurrence and expression levels of TICs in individual samples across the genome. Methods We performed single-end RNA-Seq on three human prostate adenocarcinoma samples and their corresponding normal tissues, as well as brain and universal reference samples. We developed two bioinformatics methods to specifically identify TIC events: a targeted alignment method using artificial exon-exon junctions within 200,000 bp from adjacent genes, and genomic alignment allowing splicing within individual reads. We performed further experimental verification and characterization of selected TIC and fusion events using quantitative RT-PCR and comparative genomic hybridization microarrays. Results Targeted alignment against artificial exon-exon junctions yielded 339 distinct TIC events, including 32 gene pairs with multiple isoforms. The false discovery rate was estimated to be 1.5%. Spliced alignment to the genome was less sensitive, finding only 18% of those found by targeted alignment in 33-nt reads and 59% of those in 50-nt reads. However, spliced alignment revealed 30 cases of TICs with intervening exons, in addition to distant inversions, scrambled genes, and translocations. Our findings increase the catalog of observed TIC gene pairs by 66%. We verified 6 of 6 predicted TICs in all prostate samples, and 2 of 5 predicted novel distant gene fusions, both private events among 54 prostate tumor samples tested. Expression of TICs correlates with that of the upstream gene, which can explain the prostate-specific pattern of some TIC events and the restriction of the SLC45A3-ELK4 e4-e2 TIC to ERG-negative prostate samples, as confirmed in 20 matched prostate tumor and normal samples and 9 lung cancer

  18. Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses.

    PubMed

    Borges, Aline; Tsai, Siu Mui; Caldas, Danielle Gregorio Gomes

    2012-05-01

    Selection of reference genes is an essential consideration to increase the precision and quality of relative expression analysis by the quantitative RT-PCR method. The stability of eight expressed sequence tags was evaluated to define potential reference genes to study the differential expression of common bean target genes under biotic (incompatible interaction between common bean and fungus Colletotrichum lindemuthianum) and abiotic (drought; salinity; cold temperature) stresses. The efficiency of amplification curves and quantification cycle (C (q)) were determined using LinRegPCR software. The stability of the candidate reference genes was obtained using geNorm and NormFinder software, whereas the normalization of differential expression of target genes [beta-1,3-glucanase 1 (BG1) gene for biotic stress and dehydration responsive element binding (DREB) gene for abiotic stress] was defined by REST software. High stability was obtained for insulin degrading enzyme (IDE), actin-11 (Act11), unknown 1 (Ukn1) and unknown 2 (Ukn2) genes during biotic stress, and for SKP1/ASK-interacting protein 16 (Skip16), Act11, Tubulin beta-8 (β-Tub8) and Unk1 genes under abiotic stresses. However, IDE and Act11 were indicated as the best combination of reference genes for biotic stress analysis, whereas the Skip16 and Act11 genes were the best combination to study abiotic stress. These genes should be useful in the normalization of gene expression by RT-PCR analysis in common bean, the most important edible legume.

  19. Reference Genes for Addressing Gene Expression of Bladder Cancer Cell Models under Hypoxia: A Step Towards Transcriptomic Studies

    PubMed Central

    Soares, Janine; Neves, Manuel; Santos, Lúcio Lara; Ferreira, José Alexandre

    2016-01-01

    Highly aggressive, rapidly growing tumors contain significant areas of hypoxia or anoxia as a consequence of inadequate and/or irregular blood supply. During oxygen deprivation, tumor cells withstand a panoply of adaptive responses, including a shift towards anaerobic metabolism and the reprogramming of the transcriptome. One of the major mediators of the transcriptional hypoxic response is the hypoxia-inducible factor 1 (HIF-1), whose stabilization under hypoxia acts as an oncogenic stimulus contributing to chemotherapy resistance, invasion and metastasis. Gene expression analysis by qRT-PCR is a powerful tool for cancer cells phenotypic characterization. Nevertheless, as cells undergo a severe transcriptome remodeling.in response to oxygen deficit, the precise identification of reference genes poses a significant challenge for hypoxic studies. Herein, we aim to establish the best reference genes for studying the effects of hypoxia on bladder cancer cells. Accordingly, three bladder cancer cell lines (T24, 5637, and HT1376) representative of two distinct carcinogenesis pathways to invasive cancer (FGFR3/CCND1 and E2F3/RB1) were used. Additionally, we have explored the most suitable control gene when addressing the influence of Deferoxamine Mesilate salt (DFX), an iron chelator often used to avoid the proteasomal degradation of HIF-1α, acting as an hypoxia-mimetic agent. Using bioinformatics tools (GeNorm and NormFinder), we have elected B2M and HPRT as the most stable genes for all cell lines and experimental conditions out of a panel of seven putative candidates (HPRT, ACTB, 18S, GAPDH, TBP, B2M, and SDHA). These observations set the molecular basis for future studies addressing the effect of hypoxia and particularly HIF-1α in bladder cancer cells. PMID:27835695

  20. Reference Genes for Addressing Gene Expression of Bladder Cancer Cell Models under Hypoxia: A Step Towards Transcriptomic Studies.

    PubMed

    Lima, Luís; Gaiteiro, Cristiana; Peixoto, Andreia; Soares, Janine; Neves, Manuel; Santos, Lúcio Lara; Ferreira, José Alexandre

    2016-01-01

    Highly aggressive, rapidly growing tumors contain significant areas of hypoxia or anoxia as a consequence of inadequate and/or irregular blood supply. During oxygen deprivation, tumor cells withstand a panoply of adaptive responses, including a shift towards anaerobic metabolism and the reprogramming of the transcriptome. One of the major mediators of the transcriptional hypoxic response is the hypoxia-inducible factor 1 (HIF-1), whose stabilization under hypoxia acts as an oncogenic stimulus contributing to chemotherapy resistance, invasion and metastasis. Gene expression analysis by qRT-PCR is a powerful tool for cancer cells phenotypic characterization. Nevertheless, as cells undergo a severe transcriptome remodeling.in response to oxygen deficit, the precise identification of reference genes poses a significant challenge for hypoxic studies. Herein, we aim to establish the best reference genes for studying the effects of hypoxia on bladder cancer cells. Accordingly, three bladder cancer cell lines (T24, 5637, and HT1376) representative of two distinct carcinogenesis pathways to invasive cancer (FGFR3/CCND1 and E2F3/RB1) were used. Additionally, we have explored the most suitable control gene when addressing the influence of Deferoxamine Mesilate salt (DFX), an iron chelator often used to avoid the proteasomal degradation of HIF-1α, acting as an hypoxia-mimetic agent. Using bioinformatics tools (GeNorm and NormFinder), we have elected B2M and HPRT as the most stable genes for all cell lines and experimental conditions out of a panel of seven putative candidates (HPRT, ACTB, 18S, GAPDH, TBP, B2M, and SDHA). These observations set the molecular basis for future studies addressing the effect of hypoxia and particularly HIF-1α in bladder cancer cells.

  1. Selection of Reliable Reference Genes for Gene Expression Analysis under Abiotic Stresses in the Desert Biomass Willow, Salix psammophila.

    PubMed

    Li, Jianbo; Jia, Huixia; Han, Xiaojiao; Zhang, Jin; Sun, Pei; Lu, Mengzhu; Hu, Jianjun

    2016-01-01

    Salix psammophila is a desert shrub willow that has extraordinary adaptation to abiotic stresses and plays an important role in maintaining local ecosystems. Moreover, S. psammophila is regarded as a promising biomass feedstock because of its high biomass yields and short rotation coppice cycle. However, few suitable reference genes (RGs) for quantitative real-time polymerase chain reaction (qRT-PCR) constrain the study on normalization of gene expression in S. psammophila until now. Here, we investigated the expression stabilities of 14 candidate RGs across tissue types and under four abiotic stress treatments, including heat, cold, salt, and drought treatments. After calculation of PCR efficiencies, three different software, NormFinder, geNorm, and BestKeeper were employed to analyze systematically the qRT-PCR data, and the outputs were merged by RankAggreg software. The optimal RGs selected for gene expression analysis were EF1α (Elongation factor-1 alpha) and OTU (OTU-like cysteine protease family protein) for different tissue types, UBC (Ubiquitin-conjugating enzyme E2) and LTA4H (Leukotriene A-4 hydrolase homolog) for heat treatment, HIS (Histone superfamily protein H3) and ARF2 (ADP-ribosylation factor 2) for cold treatment, OTU and ACT7 (Actin 7) for salt treatment, UBC and LTA4H for drought treatment. The expression of UBC, ARF2, and VHAC (V-type proton ATPase subunit C) varied the least across tissue types and under abiotic stresses. Furthermore, the relative genes expression profiles of one tissue-specific gene WOX1a (WUSCHEL-related homeobox 1a), and four stress-inducible genes, including Hsf-A2 (Heat shock transcription factors A2), CBF3 (C-repeat binding factor 3), HKT1 (High-Affinity K(+) Transporter 1), and GST (Glutathione S-transferase), were conducted to confirm the validity of the RGs in this study. These results provided an important RGs application guideline for gene expression characterization in S. psammophila.

  2. Identification and validation of superior reference gene for gene expression normalization via RT-qPCR in staminate and pistillate flowers of Jatropha curcas – A biodiesel plant

    PubMed Central

    Karuppaiya, Palaniyandi; Yan, Xiao-Xue; Liao, Wang; Chen, Fang; Tang, Lin

    2017-01-01

    Physic nut (Jatropha curcas L) seed oil is a natural resource for the alternative production of fossil fuel. Seed oil production is mainly depended on seed yield, which was restricted by the low ratio of staminate flowers to pistillate flowers. Further, the mechanism of physic nut flower sex differentiation has not been fully understood yet. Quantitative Real Time—Polymerase Chain Reaction is a reliable and widely used technique to quantify the gene expression pattern in biological samples. However, for accuracy of qRT-PCR, appropriate reference gene is highly desirable to quantify the target gene level. Hence, the present study was aimed to identify the stable reference genes in staminate and pistillate flowers of J. curcas. In this study, 10 candidate reference genes were selected and evaluated for their expression stability in staminate and pistillate flowers, and their stability was validated by five different algorithms (ΔCt, BestKeeper, NormFinder, GeNorm and RefFinder). Resulting, TUB and EF found to be the two most stably expressed reference for staminate flower; while GAPDH1 and EF found to be the most stably expressed reference gene for pistillate flowers. Finally, RT-qPCR assays of target gene AGAMOUS using the identified most stable reference genes confirmed the reliability of selected reference genes in different stages of flower development. AGAMOUS gene expression levels at different stages were further proved by gene copy number analysis. Therefore, the present study provides guidance for selecting appropriate reference genes for analyzing the expression pattern of floral developmental genes in staminate and pistillate flowers of J. curcas. PMID:28234941

  3. Identification and validation of superior reference gene for gene expression normalization via RT-qPCR in staminate and pistillate flowers of Jatropha curcas - A biodiesel plant.

    PubMed

    Karuppaiya, Palaniyandi; Yan, Xiao-Xue; Liao, Wang; Wu, Jun; Chen, Fang; Tang, Lin

    2017-01-01

    Physic nut (Jatropha curcas L) seed oil is a natural resource for the alternative production of fossil fuel. Seed oil production is mainly depended on seed yield, which was restricted by the low ratio of staminate flowers to pistillate flowers. Further, the mechanism of physic nut flower sex differentiation has not been fully understood yet. Quantitative Real Time-Polymerase Chain Reaction is a reliable and widely used technique to quantify the gene expression pattern in biological samples. However, for accuracy of qRT-PCR, appropriate reference gene is highly desirable to quantify the target gene level. Hence, the present study was aimed to identify the stable reference genes in staminate and pistillate flowers of J. curcas. In this study, 10 candidate reference genes were selected and evaluated for their expression stability in staminate and pistillate flowers, and their stability was validated by five different algorithms (ΔCt, BestKeeper, NormFinder, GeNorm and RefFinder). Resulting, TUB and EF found to be the two most stably expressed reference for staminate flower; while GAPDH1 and EF found to be the most stably expressed reference gene for pistillate flowers. Finally, RT-qPCR assays of target gene AGAMOUS using the identified most stable reference genes confirmed the reliability of selected reference genes in different stages of flower development. AGAMOUS gene expression levels at different stages were further proved by gene copy number analysis. Therefore, the present study provides guidance for selecting appropriate reference genes for analyzing the expression pattern of floral developmental genes in staminate and pistillate flowers of J. curcas.

  4. Renal tissue thawed for 30 minutes is still suitable for gene expression analysis.

    PubMed

    Ma, Yi; Kang, Xiao-Nan; Ding, Wen-Bin; Yang, Hao-Zheng; Wang, Ye; Zhang, Jin; Huang, Yi-Ran; Dai, Hui-Li

    2014-01-01

    Some biosamples obtained from biobanks may go through thawing before processing. We aim to evaluate the effects of thawing at room temperature for different time periods on gene expression analysis. A time course study with four time points was conducted to investigate the expression profiling on 10 thawed normal mice renal tissue samples through Affymetrix GeneChip mouse gene 2.0 st array. Microarray results were validated by quantitative real time polymerase chain reactions (qPCR) on 6 candidate reference genes and 11 target genes. Additionally, we used geNorm plus and NormFinder to identify the most stably expressed reference genes over time. The results showed RNA degraded more after longer incubation at room temperature. However, microarray results showed only 240 genes (0.91%) altered significantly in response to thawing at room temperature. The signal of majority altered probe sets decreased with thawing time, and the crossing point (Cp) values of all candidate reference genes correlated positively with the thawing time (p<0.05). The combination of B2M, ACTB and PPIA was identified as the best choice for qPCR normalization. We found most target genes were stable by using this normalization method. However, serious gene quantification errors were resulted from improper reference genes. In conclusion, thirty minutes of thawing at room temperature has a limited impact on microarray and qPCR analysis, gene expression variations due to RNA degradation in early period after thawing can be largely reduced by proper normalization.

  5. Identification of reliable reference genes for qRT-PCR studies of the developing mouse mammary gland

    PubMed Central

    van de Moosdijk, Anoeska Agatha Alida; van Amerongen, Renée

    2016-01-01

    Cell growth and differentiation are often driven by subtle changes in gene expression. Many challenges still exist in detecting these changes, particularly in the context of a complex, developing tissue. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) allows relatively high-throughput evaluation of multiple genes and developmental time points. Proper quantification of gene expression levels by qRT-PCR requires normalization to one or more reference genes. Traditionally, these genes have been selected based on their presumed “housekeeping” function, with the implicit assumption that they are stably expressed over the entire experimental set. However, this is rarely tested empirically. Here we describe the identification of novel reference genes for the mouse mammary gland based on their stable expression in published microarray datasets. We compared eight novel candidate reference genes (Arpc3, Clock, Ctbp1, Phf7, Prdx1, Sugp2, Taf11 and Usp7) to eight traditional ones (18S, Actb, Gapdh, Hmbs, Hprt, Rpl13a, Sdha and Tbp) and analysed all genes for stable expression in the mouse mammary gland from pre-puberty to adulthood using four different algorithms (GeNorm, DeltaCt, BestKeeper and NormFinder). Prdx1, Phf7 and Ctbp1 were validated as novel and reliable, tissue-specific reference genes that outperform traditional reference genes in qRT-PCR studies of postnatal mammary gland development. PMID:27752147

  6. Reference genes for quantitative real-time polymerase chain reaction studies in soybean plants under hypoxic conditions.

    PubMed

    Nakayama, T J; Rodrigues, F A; Neumaier, N; Marcelino-Guimarães, F C; Farias, J R B; de Oliveira, M C N; Borém, A; de Oliveira, A C B; Emygdio, B M; Nepomuceno, A L

    2014-02-13

    Quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful tool used to measure gene expression. However, because of its high sensitivity, the method is strongly influenced by the quality and concentration of the template cDNA and by the amplification efficiency. Relative quantification is an effective strategy for correcting random and systematic errors by using the expression level of reference gene(s) to normalize the expression level of the genes of interest. To identify soybean reference genes for use in studies of flooding stress, we compared 5 candidate reference genes (CRGs) with the NormFinder and GeNorm programs to select the best internal control. The expression stability of the CRGs was evaluated in root tissues from soybean plants subjected to hypoxic conditions. Elongation factor 1-beta and actin-11 were identified as the most appropriate genes for RT-qPCR normalization by both the NormFinder and GeNorm analyses. The expression profiles of the genes for alcohol dehydrogenase 1, sucrose synthase 4, and ascorbate peroxidase 2 were analyzed by comparing different normalizing combinations (including no normalization) of the selected reference genes. Here, we have identified potential genes for use as references for RT-qPCR normalization in experiments with soybean roots growing in O2-depleted environments, such as flooding-stressed plants.

  7. Identification of suitable reference genes for quantitative real-time PCR normalization in blotched snakehead Channa maculata.

    PubMed

    Mao, H; Chen, K; Zhu, X; Luo, Q; Zhao, J; Li, W; Wu, X; Xu, H

    2017-04-07

    A systematic study was conducted to identify reliable reference genes for normalization of gene expression analysis in the blotched snakehead Channa maculata under normal physiological conditions. Firstly, the partial complementary (c)DNA of nine candidate reference genes (actb, tmem104, ube2l3, ef1α, churc1, tmem256, rpl13a, sep15 and g6pd) were cloned from C. maculata. The expression levels of these genes were then assessed in embryos of different developmental stages and various tissue types of adult fish using quantitative real-time (qrt-)PCR. RefFinder algorithm was used to evaluate the expression stability of these genes based on their cycle-threshold (Ct ) values in the qrt-PCR analysis. Results showed that there was no single best reference gene for all stages of embryos and adult tissues tested. Furthermore, it was found that, among the nine candidate genes tested, actb and tmem104 were the most stable reference genes across adult tissue types, while sep15 and tmem256 were the most stable ones across developmental stages of embryos. These stable reference genes are recommended for normalization of gene expression analysis in C. maculata.

  8. Variation in stability of endogenous reference genes in fallopian tubes and endometrium from healthy and ectopic pregnant women.

    PubMed

    Gebeh, Alpha K; Marczylo, Emma L; Amoako, Akwasi A; Willets, Jonathon M; Konje, Justin C

    2012-01-01

    RT-qPCR is commonly employed in gene expression studies in ectopic pregnancy. Most use RN18S1, β-actin or GAPDH as internal controls without validation of their suitability as reference genes. A systematic study of the suitability of endogenous reference genes for gene expression studies in ectopic pregnancy is lacking. The aims of this study were therefore to evaluate the stability of 12 reference genes and suggest those that are stable for use as internal control genes in fallopian tubes and endometrium from ectopic pregnancy and healthy non-pregnant controls. Analysis of the results showed that the genes consistently ranked in the top six by geNorm and NormFinder algorithms, were UBC, GAPDH, CYC1 and EIF4A2 (fallopian tubes) and UBC and ATP5B (endometrium). mRNA expression of NAPE-PLD as a test gene of interest varied between the groups depending on which of the 12 reference genes was used as internal controls. This study demonstrates that arbitrary selection of reference genes for normalisation in RT-qPCR studies in ectopic pregnancy without validation, risk producing inaccurate data and should therefore be discouraged.

  9. Validation of reference genes for cryopreservation studies with the gorgonian coral endosymbiont Symbiodinium

    PubMed Central

    Chong, Gabriella; Kuo, Fu-Wen; Tsai, Sujune; Lin, Chiahsin

    2017-01-01

    Quantification by real-time RT-PCR requires a stable internal reference known as a housekeeping gene (HKG) for normalising the mRNA levels of target genes. The present study identified and validated stably expressed HKGs in post-thaw Symbiodinium clade G. Six potential HKGs, namely, pcna, gapdh, 18S rRNA, hsp90, rbcl, and ps1, were analysed using three different algorithms, namely, GeNorm, NormFinder, and BestKeeper. The GeNorm algorithm ranked the candidate genes as follows in the order of decreasing stability: pcna and gapdh > ps1 > 18S rRNA > hsp90 > rbcl. Results obtained using the NormFinder algorithm also showed that pcna was the most stable HKG and ps1 was the second most stable HKG. We found that the candidate HKGs examined in this study showed variable stability with respect to the three algorithms. These results indicated that both pcna and ps1 were suitable for normalising target gene expression determined by performing real-time RT-PCR in cryopreservation studies on Symbiodinium clade G. The results of the present study would help future studies to elucidate the effect of cryopreservation on gene expression in dinoflagellates. PMID:28067273

  10. Identification and Validation of Reference Genes for the Normalization of Gene Expression Data in qRT-PCR Analysis in Aphis gossypii (Hemiptera: Aphididae).

    PubMed

    Ma, Kang-Sheng; Li, Fen; Liang, Ping-Zhuo; Chen, Xue-Wei; Liu, Ying; Gao, Xi-Wu

    2016-01-01

    To obtain accurate and reliable results from quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis, it is necessary to select suitable reference genes as standards for normalizing target gene expression data. QRT-PCR is a popular analytical methodology for studying gene expression and it has been used widely in studies of Aphis gossypii Glover in recent years. However, there is absence of study on the stability of the expression of reference genes in A. gossypii. In this study, eight commonly used candidate reference genes, including 18S, 28S, β-ACT, GAPDH, EF1α, RPL7, α-TUB, and TBP, were evaluated under various experimental conditions to assess their suitability for use in the normalization of qRT-PCR data. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated by performing normalizations of expression data for the HSP70 gene. The results showed the most suitable combinations of reference genes for the different experimental conditions. For experiments based on divergent developmental stages, EF1α, β-ACT, and RPL7 are the optimal reference gene combination, both EF1α and β-ACT are the optimal combination used in the experiments of different geographical populations, whereas for experiments of the temperature changes, the combination of GAPDH and RPL7 is optimal, both 18S and β-ACT are an optimal combination for feeding assay experiments. These research results should be useful for the selection of the suitable reference genes to obtain reliable qRT-PCR data in the gene expression study of A. gossypii.

  11. Evaluation of Reference Genes for RT-qPCR Studies in the Seagrass Zostera muelleri Exposed to Light Limitation

    PubMed Central

    Schliep, M.; Pernice, M.; Sinutok, S.; Bryant, C. V.; York, P. H.; Rasheed, M. A.; Ralph, P. J.

    2015-01-01

    Seagrass meadows are threatened by coastal development and global change. In the face of these pressures, molecular techniques such as reverse transcription quantitative real-time PCR (RT-qPCR) have great potential to improve management of these ecosystems by allowing early detection of chronic stress. In RT-qPCR, the expression levels of target genes are estimated on the basis of reference genes, in order to control for RNA variations. Although determination of suitable reference genes is critical for RT-qPCR studies, reports on the evaluation of reference genes are still absent for the major Australian species Zostera muelleri subsp. capricorni (Z. muelleri). Here, we used three different software (geNorm, NormFinder and Bestkeeper) to evaluate ten widely used reference genes according to their expression stability in Z. muelleri exposed to light limitation. We then combined results from different software and used a consensus rank of four best reference genes to validate regulation in Photosystem I reaction center subunit IV B and Heat Stress Transcription factor A- gene expression in Z. muelleri under light limitation. This study provides the first comprehensive list of reference genes in Z. muelleri and demonstrates RT-qPCR as an effective tool to identify early responses to light limitation in seagrass. PMID:26592440

  12. Selection of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in Brassica napus under various stress conditions.

    PubMed

    Wang, Zheng; Chen, Yu; Fang, Hedi; Shi, Haifeng; Chen, Keping; Zhang, Zhiyan; Tan, Xiaoli

    2014-10-01

    Data normalization is essential for reliable output of quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) assays, as the unsuitable choice of reference gene(s), whose expression might be influenced by exogenous treatments in plant tissues, could cause misinterpretation of results. To date, no systematic studies on reference genes have been performed in stressed Brassica napus. In this study, we investigated the expression variations of nine candidate reference genes in 40 samples of B. napus leaves subjected to various exogenous treatments. Parallel analyses by geNorm and NormFinder revealed that optimal reference genes differed across the different sets of samples. The best-ranked reference genes were PP2A and TIP41 for salt stress, TIP41 and ACT7 for heavy metal (Cr(6+)) stress, PP2A and UBC21 for drought stress, F-box and SAND for cold stress, F-box and ZNF for salicylic acid stress, TIP41, ACT7, and PP2A for methyl jasmonate stress, TIP41 and ACT7 for abscisic acid stress, and TIP41, UBC21, and PP2A for Sclerotinia sclerotiorum stress. Two newly employed reference genes, TIP41 and PP2A, showed better performances, suggesting their suitability in multiple conditions. To further validate the suitability of the reference genes, the expression patterns of BnWRKY40 and BnMKS1 were studied in parallel. This study is the first systematic analysis of reference gene selection for qRT-PCR normalization in B. napus, an agriculturally important crop, under different stress conditions. The results will contribute toward more accurate and widespread use of qRT-PCR in gene analysis of the genus Brassica.

  13. Reference genes for quantitative real-time PCR analysis in symbiont Entomomyces delphacidicola of Nilaparvata lugens (Stål)

    PubMed Central

    Wan, Pin-Jun; Tang, Yao-Hua; Yuan, San-Yue; He, Jia-Chun; Wang, Wei-Xia; Lai, Feng-Xiang; Fu, Qiang

    2017-01-01

    Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) is a major rice pest that harbors an endosymbiont ascomycete fungus, Entomomyces delphacidicola str. NLU (also known as yeast-like symbiont, YLS). Driving by demand of novel population management tactics (e.g. RNAi), the importance of YLS has been studied and revealed, which greatly boosts the interest of molecular level studies related to YLS. The current study focuses on reference genes for RT-qPCR studies related to YLS. Eight previously unreported YLS genes were cloned, and their expressions were evaluated for N. lugens samples of different developmental stages and sexes, and under different nutritional conditions and temperatures. Expression stabilities were analyzed by BestKeeper, geNorm, NormFinder, ΔCt method and RefFinder. Furthermore, the selected reference genes for RT-qPCR of YLS genes were validated using targeted YLS genes that respond to different nutritional conditions (amino acid deprivation) and RNAi. The results suggest that ylsRPS15p/ylsACT are the most suitable reference genes for temporal gene expression profiling, while ylsTUB/ylsACT and ylsRPS15e/ylsGADPH are the most suitable reference gene choices for evaluating nutrition and temperature effects. Validation studies demonstrated the advantage of using endogenous YLS reference genes for YLS studies. PMID:28198810

  14. Creating reference gene annotation for the mouse C57BL6/J genome assembly.

    PubMed

    Mudge, Jonathan M; Harrow, Jennifer

    2015-10-01

    Annotation on the reference genome of the C57BL6/J mouse has been an ongoing project ever since the draft genome was first published. Initially, the principle focus was on the identification of all protein-coding genes, although today the importance of describing long non-coding RNAs, small RNAs, and pseudogenes is recognized. Here, we describe the progress of the GENCODE mouse annotation project, which combines manual annotation from the HAVANA group with Ensembl computational annotation, alongside experimental and in silico validation pipelines from other members of the consortium. We discuss the more recent incorporation of next-generation sequencing datasets into this workflow, including the usage of mass-spectrometry data to potentially identify novel protein-coding genes. Finally, we will outline how the C57BL6/J genebuild can be used to gain insights into the variant sites that distinguish different mouse strains and species.

  15. Assessment of brain reference genes for RT-qPCR studies in neurodegenerative diseases

    PubMed Central

    Rydbirk, Rasmus; Folke, Jonas; Winge, Kristian; Aznar, Susana; Pakkenberg, Bente; Brudek, Tomasz

    2016-01-01

    Evaluation of gene expression levels by reverse transcription quantitative real-time PCR (RT-qPCR) has for many years been the favourite approach for discovering disease-associated alterations. Normalization of results to stably expressed reference genes (RGs) is pivotal to obtain reliable results. This is especially important in relation to neurodegenerative diseases where disease-related structural changes may affect the most commonly used RGs. We analysed 15 candidate RGs in 98 brain samples from two brain regions from Alzheimer’s disease (AD), Parkinson’s disease (PD), Multiple System Atrophy, and Progressive Supranuclear Palsy patients. Using RefFinder, a web-based tool for evaluating RG stability, we identified the most stable RGs to be UBE2D2, CYC1, and RPL13 which we recommend for future RT-qPCR studies on human brain tissue from these patients. None of the investigated genes were affected by experimental variables such as RIN, PMI, or age. Findings were further validated by expression analyses of a target gene GSK3B, known to be affected by AD and PD. We obtained high variations in GSK3B levels when contrasting the results using different sets of common RG underlining the importance of a priori validation of RGs for RT-qPCR studies. PMID:27853238

  16. A combined strategy of "in silico" transcriptome analysis and web search engine optimization allows an agile identification of reference genes suitable for normalization in gene expression studies.

    PubMed

    Faccioli, Primetta; Ciceri, Gian Paolo; Provero, Paolo; Stanca, Antonio Michele; Morcia, Caterina; Terzi, Valeria

    2007-03-01

    Traditionally housekeeping genes have been employed as endogenous reference (internal control) genes for normalization in gene expression studies. Since the utilization of single housekeepers cannot assure an unbiased result, new normalization methods involving multiple housekeeping genes and normalizing using their mean expression have been recently proposed. Moreover, since a gold standard gene suitable for every experimental condition does not exist, it is also necessary to validate the expression stability of every putative control gene on the specific requirements of the planned experiment. As a consequence, finding a good set of reference genes is for sure a non-trivial problem requiring quite a lot of lab-based experimental testing. In this work we identified novel candidate barley reference genes suitable for normalization in gene expression studies. An advanced web search approach aimed to collect, from publicly available web resources, the most interesting information regarding the expression profiling of candidate housekeepers on a specific experimental basis has been set up and applied, as an example, on stress conditions. A complementary lab-based analysis has been carried out to verify the expression profile of the selected genes in different tissues and during heat shock response. This combined dry/wet approach can be applied to any species and physiological condition of interest and can be considered very helpful to identify putative reference genes to be shortlisted every time a new experimental design has to be set up.

  17. Different distribution patterns of ten virulence genes in Legionella reference strains and strains isolated from environmental water and patients.

    PubMed

    Zhan, Xiao-Yong; Hu, Chao-Hui; Zhu, Qing-Yi

    2016-04-01

    Virulence genes are distinct regions of DNA which are present in the genome of pathogenic bacteria and absent in nonpathogenic strains of the same or related species. Virulence genes are frequently associated with bacterial pathogenicity in genus Legionella. In the present study, an assay was performed to detect ten virulence genes, including iraA, iraB, lvrA, lvrB, lvhD, cpxR, cpxA, dotA, icmC and icmD in different pathogenicity islands of 47 Legionella reference strains, 235 environmental strains isolated from water, and 4 clinical strains isolated from the lung tissue of pneumonia patients. The distribution frequencies of these genes in reference or/and environmental L. pneumophila strains were much higher than those in reference non-L. pneumophila or/and environmental non-L. pneumophila strains, respectively. L. pneumophila clinical strains also maintained higher frequencies of these genes compared to four other types of Legionella strains. Distribution frequencies of these genes in reference L. pneumophila strains were similar to those in environmental L. pneumophila strains. In contrast, environmental non-L. pneumophila maintained higher frequencies of these genes compared to those found in reference non-L. pneumophila strains. This study illustrates the association of virulence genes with Legionella pathogenicity and reveals the possible virulence evolution of non-L. pneumophia strains isolated from environmental water.

  18. Validation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in strawberry fruits using different cultivars and osmotic stresses.

    PubMed

    Galli, Vanessa; Borowski, Joyce Moura; Perin, Ellen Cristina; Messias, Rafael da Silva; Labonde, Julia; Pereira, Ivan dos Santos; Silva, Sérgio Delmar Dos Anjos; Rombaldi, Cesar Valmor

    2015-01-10

    The increasing demand of strawberry (Fragaria×ananassa Duch) fruits is associated mainly with their sensorial characteristics and the content of antioxidant compounds. Nevertheless, the strawberry production has been hampered due to its sensitivity to abiotic stresses. Therefore, to understand the molecular mechanisms highlighting stress response is of great importance to enable genetic engineering approaches aiming to improve strawberry tolerance. However, the study of expression of genes in strawberry requires the use of suitable reference genes. In the present study, seven traditional and novel candidate reference genes were evaluated for transcript normalization in fruits of ten strawberry cultivars and two abiotic stresses, using RefFinder, which integrates the four major currently available software programs: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. The results indicate that the expression stability is dependent on the experimental conditions. The candidate reference gene DBP (DNA binding protein) was considered the most suitable to normalize expression data in samples of strawberry cultivars and under drought stress condition, and the candidate reference gene HISTH4 (histone H4) was the most stable under osmotic stresses and salt stress. The traditional genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 18S (18S ribosomal RNA) were considered the most unstable genes in all conditions. The expression of phenylalanine ammonia lyase (PAL) and 9-cis epoxycarotenoid dioxygenase (NCED1) genes were used to further confirm the validated candidate reference genes, showing that the use of an inappropriate reference gene may induce erroneous results. This study is the first survey on the stability of reference genes in strawberry cultivars and osmotic stresses and provides guidelines to obtain more accurate RT-qPCR results for future breeding efforts.

  19. Identification of Importin 8 (IPO8) as the most accurate reference gene for the clinicopathological analysis of lung specimens

    PubMed Central

    Nguewa, Paul A; Agorreta, Jackeline; Blanco, David; Lozano, Maria Dolores; Gomez-Roman, Javier; Sanchez, Blas A; Valles, Iñaki; Pajares, Maria J; Pio, Ruben; Rodriguez, Maria Jose; Montuenga, Luis M; Calvo, Alfonso

    2008-01-01

    Background The accurate normalization of differentially expressed genes in lung cancer is essential for the identification of novel therapeutic targets and biomarkers by real time RT-PCR and microarrays. Although classical "housekeeping" genes, such as GAPDH, HPRT1, and beta-actin have been widely used in the past, their accuracy as reference genes for lung tissues has not been proven. Results We have conducted a thorough analysis of a panel of 16 candidate reference genes for lung specimens and lung cell lines. Gene expression was measured by quantitative real time RT-PCR and expression stability was analyzed with the softwares GeNorm and NormFinder, mean of |ΔCt| (= |Ct Normal-Ct tumor|) ± SEM, and correlation coefficients among genes. Systematic comparison between candidates led us to the identification of a subset of suitable reference genes for clinical samples: IPO8, ACTB, POLR2A, 18S, and PPIA. Further analysis showed that IPO8 had a very low mean of |ΔCt| (0.70 ± 0.09), with no statistically significant differences between normal and malignant samples and with excellent expression stability. Conclusion Our data show that IPO8 is the most accurate reference gene for clinical lung specimens. In addition, we demonstrate that the commonly used genes GAPDH and HPRT1 are inappropriate to normalize data derived from lung biopsies, although they are suitable as reference genes for lung cell lines. We thus propose IPO8 as a novel reference gene for lung cancer samples. PMID:19014639

  20. Selection of Reliable Reference Genes for Gene Expression Analysis under Abiotic Stresses in the Desert Biomass Willow, Salix psammophila

    PubMed Central

    Li, Jianbo; Jia, Huixia; Han, Xiaojiao; Zhang, Jin; Sun, Pei; Lu, Mengzhu; Hu, Jianjun

    2016-01-01

    Salix psammophila is a desert shrub willow that has extraordinary adaptation to abiotic stresses and plays an important role in maintaining local ecosystems. Moreover, S. psammophila is regarded as a promising biomass feedstock because of its high biomass yields and short rotation coppice cycle. However, few suitable reference genes (RGs) for quantitative real-time polymerase chain reaction (qRT-PCR) constrain the study on normalization of gene expression in S. psammophila until now. Here, we investigated the expression stabilities of 14 candidate RGs across tissue types and under four abiotic stress treatments, including heat, cold, salt, and drought treatments. After calculation of PCR efficiencies, three different software, NormFinder, geNorm, and BestKeeper were employed to analyze systematically the qRT-PCR data, and the outputs were merged by RankAggreg software. The optimal RGs selected for gene expression analysis were EF1α (Elongation factor-1 alpha) and OTU (OTU-like cysteine protease family protein) for different tissue types, UBC (Ubiquitin-conjugating enzyme E2) and LTA4H (Leukotriene A-4 hydrolase homolog) for heat treatment, HIS (Histone superfamily protein H3) and ARF2 (ADP-ribosylation factor 2) for cold treatment, OTU and ACT7 (Actin 7) for salt treatment, UBC and LTA4H for drought treatment. The expression of UBC, ARF2, and VHAC (V-type proton ATPase subunit C) varied the least across tissue types and under abiotic stresses. Furthermore, the relative genes expression profiles of one tissue-specific gene WOX1a (WUSCHEL-related homeobox 1a), and four stress-inducible genes, including Hsf-A2 (Heat shock transcription factors A2), CBF3 (C-repeat binding factor 3), HKT1 (High-Affinity K+ Transporter 1), and GST (Glutathione S-transferase), were conducted to confirm the validity of the RGs in this study. These results provided an important RGs application guideline for gene expression characterization in S. psammophila. PMID:27761137

  1. Methods of RNA preparation affect mRNA abundance quantification of reference genes in pig maturing oocytes.

    PubMed

    Wang, Y-K; Li, X; Song, Z-Q; Yang, C-X

    2017-04-13

    To ensure accurate normalization and quantification of target RNA transcripts using reverse transcription quantitative polymerase chain reaction (RT-qPCR), most studies focus on the identification of stably expressed gene(s) as internal reference. However, RNA preparation methods could also be an important factor, especially for test samples of limited quantity (e.g. oocytes). In this study, we aimed to select appropriate reference gene(s), and evaluate the effect of RNA preparation methods on gene expression quantification in porcine oocytes and cumulus cells during in vitro maturation. Expression profiles of seven genes (GAPDH, 18S, YWHAG, BACT, RPL4, HPRT1 and PPIA) were examined, on RNA samples extracted from cumulus cells (RNeasy Kit) and oocytes (RNeasy Kit and Lysis Kit) during in vitro maturation, respectively. Interestingly, different RNA preparation methods were found to potentially affect the quantification of reference gene expression in pig oocytes cultured in vitro. After geNorm analyses, the most suitable genes for normalization were identified, GAPDH/18S for cumulus cells and YWHAG/BACT for oocytes, respectively. Thus, our results provide useful data and information on the selection of better reference genes and RNA preparation method for related functional studies.

  2. Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.

    PubMed

    Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

    2013-01-01

    Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.

  3. Functional classification of genes using semantic distance and fuzzy clustering approach: evaluation with reference sets and overlap analysis.

    PubMed

    Devignes, Marie-Dominique; Benabderrahmane, Sidahmed; Smaïl-Tabbone, Malika; Napoli, Amedeo; Poch, Olivier

    2012-01-01

    Functional classification aims at grouping genes according to their molecular function or the biological process they participate in. Evaluating the validity of such unsupervised gene classification remains a challenge given the variety of distance measures and classification algorithms that can be used. We evaluate here functional classification of genes with the help of reference sets: KEGG (Kyoto Encyclopaedia of Genes and Genomes) pathways and Pfam clans. These sets represent ground truth for any distance based on GO (Gene Ontology) biological process and molecular function annotations respectively. Overlaps between clusters and reference sets are estimated by the F-score method. We test our previously described IntelliGO semantic distance with hierarchical and fuzzy C-means clustering and we compare results with the state-of-the-art DAVID (Database for Annotation Visualisation and Integrated Discovery) functional classification method. Finally, study of best matching clusters to reference sets leads us to propose a set-difference method for discovering missing information.

  4. SHORT-COMMUNICATION Validation of reference genes for real-time quantitative PCR in tambaqui (Colossoma macropomum).

    PubMed

    Nascimento, A R; Silva, G F; Gualberto, G F; Almeida, F L

    2016-12-23

    Tambaqui, Colossoma macropomum, is the main native freshwater fish in Brazilian aquaculture. Therefore, intensive research pressure has been applied to the species to support new technologies for tambaqui farming. Molecular biology represents a tool that can be used to investigate every field of applied biology, from fish physiology to the effects of climate change. Based on the importance of reference genes for the relative or absolute quantification of gene transcripts, we cloned and sequenced three candidate reference genes in tambaqui (18S ribossomal RNA - 18s, glyceraldehyde-3-phosphate dehydrogenase - gapdh, and actin beta - β-actin), and validated a set of primers for each gene for use in real-time quantitative PCR. The results were evaluated by RefFinder, which indicated that β-actin is the most suitable reference gene for tambaqui among those studied, followed by 18s.

  5. Evaluation of microbial qPCR workflows using engineered Saccharomyces cerevisiae

    PubMed Central

    Da Silva, S.M.; Vang, L.K.; Olson, N.D.; Lund, S.P.; Downey, A.S.; Kelman, Z.; Salit, M.L.; Lin, N.J.; Morrow, J.B.

    2016-01-01

    Aims We describe the development and interlaboratory study of modified Saccharomyces cerevisiae as a candidate material to evaluate a full detection workflow including DNA extraction and quantitative polymerase chain reaction (qPCR). Methods and results S. cerevisiae NE095 was prepared by stable insertion of DNA sequence External RNA Control Consortium-00095 into S. cerevisiae BY4739 to convey selectivity. For the interlaboratory study, a binomial regression model was used to select three cell concentrations, high (4 × 107 cells ml−1), intermediate (4 × 105 cells ml−1) and low (4 × 103 cells ml−1), and the number of samples per concentration. Seven participants, including potential end users, had combined rates of positive qPCR detection (quantification cycle <37) of 100%, 40%, and 0% for high, intermediate, and low concentrations, respectively. Conclusions The NE095 strain was successfully detected by all participants, with the high concentration indicating a potential target concentration for a reference material. Significance and impact of the study The engineered yeast has potential to support measurement assurance for the analytical process of qPCR, encompassing the method, equipment, and operator, to increase confidence in results and better inform decision-making in areas of applied microbiology. This material can also support process assessment for other DNA-based detection technologies. PMID:27077050

  6. Selection and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) under Heat and Salt Stress Conditions

    PubMed Central

    Sinha, Pallavi; Saxena, Rachit K.; Singh, Vikas K.; Krishnamurthy, L.; Varshney, Rajeev K.

    2015-01-01

    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. PMID:27242803

  7. Identifying Stable Reference Genes for qRT-PCR Normalisation in Gene Expression Studies of Narrow-Leafed Lupin (Lupinus angustifolius L.).

    PubMed

    Taylor, Candy M; Jost, Ricarda; Erskine, William; Nelson, Matthew N

    2016-01-01

    Quantitative Reverse Transcription PCR (qRT-PCR) is currently one of the most popular, high-throughput and sensitive technologies available for quantifying gene expression. Its accurate application depends heavily upon normalisation of gene-of-interest data with reference genes that are uniformly expressed under experimental conditions. The aim of this study was to provide the first validation of reference genes for Lupinus angustifolius (narrow-leafed lupin, a significant grain legume crop) using a selection of seven genes previously trialed as reference genes for the model legume, Medicago truncatula. In a preliminary evaluation, the seven candidate reference genes were assessed on the basis of primer specificity for their respective targeted region, PCR amplification efficiency, and ability to discriminate between cDNA and gDNA. Following this assessment, expression of the three most promising candidates [Ubiquitin C (UBC), Helicase (HEL), and Polypyrimidine tract-binding protein (PTB)] was evaluated using the NormFinder and RefFinder statistical algorithms in two narrow-leafed lupin lines, both with and without vernalisation treatment, and across seven organ types (cotyledons, stem, leaves, shoot apical meristem, flowers, pods and roots) encompassing three developmental stages. UBC was consistently identified as the most stable candidate and has sufficiently uniform expression that it may be used as a sole reference gene under the experimental conditions tested here. However, as organ type and developmental stage were associated with greater variability in relative expression, it is recommended using UBC and HEL as a pair to achieve optimal normalisation. These results highlight the importance of rigorously assessing candidate reference genes for each species across a diverse range of organs and developmental stages. With emerging technologies, such as RNAseq, and the completion of valuable transcriptome data sets, it is possible that other potentially more

  8. Reference genes for QRT-PCR tested under various stress conditions in Folsomia candida and Orchesella cincta (Insecta, Collembola)

    PubMed Central

    de Boer, Muriel E; de Boer, Tjalf E; Mariën, Janine; Timmermans, Martijn JTN; Nota, Benjamin; van Straalen, Nico M; Ellers, Jacintha; Roelofs, Dick

    2009-01-01

    Background Genomic studies measuring transcriptional responses to changing environments and stress currently make their way into the field of evolutionary ecology and ecotoxicology. To investigate a small to medium number of genes or to confirm large scale microarray studies, Quantitative Reverse Transcriptase PCR (QRT-PCR) can achieve high accuracy of quantification when key standards, such as normalization, are carefully set. In this study, we validated potential reference genes for their use as endogenous controls under different chemical and physical stresses in two species of soil-living Collembola, Folsomia candida and Orchesella cincta. Treatments for F. candida were cadmium exposure, phenanthrene exposure, desiccation, heat shock and pH stress, and for O. cincta cadmium, desiccation, heat shock and starvation. Results Eight potential reference genes for F. candida and seven for O. cincta were ranked by their stability per stress factor using the programs geNorm and Normfinder. For F. candida the succinate dehydrogenase (SDHA) and eukaryotic transcription initiation factor 1A (ETIF) genes were found the most stable over the different treatments, while for O. cincta, the beta actin (ACTb) and tyrosine 3-monooxygenase (YWHAZ) genes were the most stable. Conclusion We present a panel of reference genes for two emerging ecological genomic model species tested under a variety of treatments. Within each species, different treatments resulted in differences in the top stable reference genes. Moreover, the two species differed in suitable reference genes even when exposed to similar stresses. This might be attributed to dissimilarity of physiology. It is vital to rigorously test a panel of reference genes for each species and treatment, in advance of relative quantification of QRT-PCR gene expression measurements. PMID:19486513

  9. Identification and validation of reference genes for quantitative real-time PCR studies in long yellow daylily, Hemerocallis citrina Borani

    PubMed Central

    Wang, Jinyao; Kang, Xiuping; Weng, Yiqun

    2017-01-01

    Gene expression analysis using reverse transcription quantitative real-time PCR (RT-qPCR) requires the use of reference gene(s) in the target species. The long yellow daylily, Hemerocallis citrina Baroni. is rich in beneficial secondary metabolites and is considered as a functional vegetable. It is widely cultivated and consumed in East Asian countries. However, reference genes for use in RT-qPCR in H. citrina are not available. In the present study, six potential reference genes, actin (ACT), AP-4 complex subunit (AP4), tubulin (TUB), ubiquitin (UBQ), 18S and 60S ribosomal RNA, were selected and their expression stability in different developmental stages, organs and accessions was evaluated using four statistical software packages (geNorm, NormFinder, BestKeeper, and RefFinder). For commercial flower buds of different landraces, the combination of 60S, TUB, and AP4 was appropriate whereas ACT and 60S was suitable for normalization of different organs. In addition, AP4 exhibited the most stable expression in flower buds among different developmental stages. UBQ was less stable than the other reference genes under the experimental conditions except under different organs was 18S. The relative expression levels of two genes, primary-amine oxidase (HcAOC3) and tyrosine aminotransferase (HcTAT) which play important roles in alkaloid biosynthesis were also examined in different organs of the ‘Datong’ landrace, which further confirmed the results of selected reference genes. This is the first report to evaluate the stability of reference genes in the long yellow daylily that can serve as a foundation for RT-qPCR analysis of gene expression in this species. PMID:28362875

  10. Identification of Suitable Reference Genes for Investigating Gene Expression in Anterior Cruciate Ligament Injury by Using Reverse Transcription-Quantitative PCR.

    PubMed

    Leal, Mariana Ferreira; Astur, Diego Costa; Debieux, Pedro; Arliani, Gustavo Gonçalves; Silveira Franciozi, Carlos Eduardo; Loyola, Leonor Casilla; Andreoli, Carlos Vicente; Smith, Marília Cardoso; Pochini, Alberto de Castro; Ejnisman, Benno; Cohen, Moises

    2015-01-01

    The anterior cruciate ligament (ACL) is one of the most frequently injured structures during high-impact sporting activities. Gene expression analysis may be a useful tool for understanding ACL tears and healing failure. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has emerged as an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. Here, we evaluated the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1, and TBP) by using ACL samples of 39 individuals with ACL tears (20 with isolated ACL tears and 19 with ACL tear and combined meniscal injury) and of 13 controls. The stability of the candidate reference genes was determined by using the NormFinder, geNorm, BestKeeper DataAssist, and RefFinder software packages and the comparative ΔCt method. ACTB was the best single reference gene and ACTB+TBP was the best gene pair. The GenEx software showed that the accumulated standard deviation is reduced when a larger number of reference genes is used for gene expression normalization. However, the use of a single reference gene may not be suitable. To identify the optimal combination of reference genes, we evaluated the expression of FN1 and PLOD1. We observed that at least 3 reference genes should be used. ACTB+HPRT1+18S is the best trio for the analyses involving isolated ACL tears and controls. Conversely, ACTB+TBP+18S is the best trio for the analyses involving (1) injured ACL tears and controls, and (2) ACL tears of patients with meniscal tears and controls. Therefore, if the gene expression study aims to compare non-injured ACL, isolated ACL tears and ACL tears from patients with meniscal tear as three independent groups ACTB+TBP+18S+HPRT1 should be used. In conclusion, 3 or more genes should be used as reference genes for analysis of ACL samples of individuals with and without ACL tears.

  11. Validation of Reference Genes for RT-qPCR Studies of Gene Expression in Preharvest and Postharvest Longan Fruits under Different Experimental Conditions

    PubMed Central

    Wu, Jianyang; Zhang, Hongna; Liu, Liqin; Li, Weicai; Wei, Yongzan; Shi, Shengyou

    2016-01-01

    Reverse transcription quantitative PCR (RT-qPCR) as the accurate and sensitive method is use for gene expression analysis, but the veracity and reliability result depends on whether select appropriate reference gene or not. To date, several reliable reference gene validations have been reported in fruits trees, but none have been done on preharvest and postharvest longan fruits. In this study, 12 candidate reference genes, namely, CYP, RPL, GAPDH, TUA, TUB, Fe-SOD, Mn-SOD, Cu/Zn-SOD, 18SrRNA, Actin, Histone H3, and EF-1a, were selected. Expression stability of these genes in 150 longan samples was evaluated and analyzed using geNorm and NormFinder algorithms. Preharvest samples consisted of seven experimental sets, including different developmental stages, organs, hormone stimuli (NAA, 2,4-D, and ethephon) and abiotic stresses (bagging and girdling with defoliation). Postharvest samples consisted of different temperature treatments (4 and 22°C) and varieties. Our findings indicate that appropriate reference gene(s) should be picked for each experimental condition. Our data further showed that the commonly used reference gene Actin does not exhibit stable expression across experimental conditions in longan. Expression levels of the DlACO gene, which is a key gene involved in regulating fruit abscission under girdling with defoliation treatment, was evaluated to validate our findings. In conclusion, our data provide a useful framework for choice of suitable reference genes across different experimental conditions for RT-qPCR analysis of preharvest and postharvest longan fruits. PMID:27375640

  12. Evaluation of Reference Genes for RT-qPCR in Tribolium castaneum (Coleoptera: Tenebrionidae) Under UVB Stress.

    PubMed

    Sang, Wen; He, Li; Wang, Xiao-Ping; Zhu-Salzman, Keyan; Lei, Chao-Liang

    2015-04-01

    Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) has become a widely used technique to quantify gene expression. It is necessary to select appropriate reference genes for normalization. In the present study, we assessed the expression stability of seven candidate genes in Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) irradiated by ultraviolet B (UVB) at different developmental stages for various irradiation time periods. The algorithms of geNorm, NormFinder, and BestKeeper were applied to determine the stability of these candidate genes. Ribosomal protein genes RpS3, RpL13A, and β-actin gene (ActB) showed the highest stability across all UVB irradiation time points, whereas expression of other normally used reference genes, such as those encoding the β-tubulin gene TUBB and the E-cadherin gene CAD, varied at different developmental stages. This study will potentially provide more suitable reference gene candidates for RT-qPCR analysis in T. castaneum subjected to environmental stresses, particularly UV irradiation.

  13. Potential Risks in the Paradigm of Basic to Translational Research: A Critical Evaluation of qPCR Telomere Size Techniques

    PubMed Central

    Lustig, Arthur J

    2015-01-01

    Real time qPCR has become the method of choice for rapid large-scale telomere length measurements. Large samples sizes are critical for clinical trials, and epidemiological studies. QPCR has become such routine procedure that it is often used with little critical analysis. With proper controls, the mean telomere size can be derived from the data and even the size can be estimated. But there is a need for more consistent and reliable controls that will provide closer to the actual mean size can be obtained with uniform consensus controls. Although originating at the level of basic telomere research, many researchers less familiar with telomeres often misunderstand the source and significance of the qPCR metric. These include researchers and clinicians who are interested in having a rapid tool to produce exciting results in disease prognostics and diagnostics than in the multiple characteristics of telomeres that form the basis of the measurement. But other characteristics of the non-bimodal and heterogeneous telomeres as well as the complexities of telomere dynamics are not easily related to qPCR mean telomere values. The qPCR metric does not reveal the heterogeneity and dynamics of telomeres. This is a critical issue since mutations in multiple genes including telomerase can cause telomere dysfunction and a loss of repeats. The smallest cellular telomere has been shown to arrest growth of the cell carrying the dysfunction telomere. A goal for the future is a simple method that takes into account the heterogeneity by measuring the highest and lowest values as part of the scheme to compare. In the absence of this technique, Southern blots need to be performed in a subset of qPCR samples for both mean telomere size and the upper and lower extremes of the distribution. Most importantly, there is a need for greater transparency in discussing the limitations of the qPCR data. Given the potentially exciting qPCR telomere size results emerging from clinical studies that

  14. Selection and evaluation of new reference genes for RT-qPCR analysis in Epinephelus akaara based on transcriptome data.

    PubMed

    Wang, Huan; Zhang, Xiang; Liu, Qiaohong; Liu, Xiaochun; Ding, Shaoxiong

    2017-01-01

    Groupers are an economically important fish species in world fishery markets. Because many studies using RT-qPCR have addressed gene expression in groupers, appropriate reference genes are required to obtain reliable and accurate results. In this study, the most suitable reference genes were identified from eleven candidate genes of one of the most valuable species, Epinephelus akaara, in a range of different experimental conditions. Using the software packages geNorm, NormFinder, BestKeeper and refFinder, three traditionally used reference genes, β-actin (β-ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-2-microglobulin (B2M), were identified as not suitable for E. akaara gene expression studies, whereas two newly identified reference genes, conserved oligomeric Golgi complex subunit 5 (Cog5) and brefeldin a-inhibited guanine nucleotide-exchange protein 1 (ARFGEF1), could be universally applied under all the tested conditions. These data provide the foundation for more precise results in RT-qPCR studies of gene expression in E. akaara and other Epinephelus species.

  15. Transcriptome-wide identification of reference genes for expression analysis of soybean responses to drought stress along the day

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The soybean transcriptome displays strong variation along the day in optimal growth conditions and also in response to adverse circumstances, like drought stress. However, no study conducted to date has presented suitable reference genes, with stable expression along the day, for relative gene expre...

  16. Selection and evaluation of new reference genes for RT-qPCR analysis in Epinephelus akaara based on transcriptome data

    PubMed Central

    Wang, Huan; Zhang, Xiang; Liu, Qiaohong; Liu, Xiaochun; Ding, Shaoxiong

    2017-01-01

    Groupers are an economically important fish species in world fishery markets. Because many studies using RT-qPCR have addressed gene expression in groupers, appropriate reference genes are required to obtain reliable and accurate results. In this study, the most suitable reference genes were identified from eleven candidate genes of one of the most valuable species, Epinephelus akaara, in a range of different experimental conditions. Using the software packages geNorm, NormFinder, BestKeeper and refFinder, three traditionally used reference genes, β-actin (β-ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-2-microglobulin (B2M), were identified as not suitable for E. akaara gene expression studies, whereas two newly identified reference genes, conserved oligomeric Golgi complex subunit 5 (Cog5) and brefeldin a-inhibited guanine nucleotide-exchange protein 1 (ARFGEF1), could be universally applied under all the tested conditions. These data provide the foundation for more precise results in RT-qPCR studies of gene expression in E. akaara and other Epinephelus species. PMID:28182746

  17. Transcriptome-Wide Identification of Reference Genes for Expression Analysis of Soybean Responses to Drought Stress along the Day.

    PubMed

    Marcolino-Gomes, Juliana; Rodrigues, Fabiana Aparecida; Fuganti-Pagliarini, Renata; Nakayama, Thiago Jonas; Ribeiro Reis, Rafaela; Bouças Farias, Jose Renato; Harmon, Frank G; Correa Molinari, Hugo Bruno; Correa Molinari, Mayla Daiane; Nepomuceno, Alexandre

    2015-01-01

    The soybean transcriptome displays strong variation along the day in optimal growth conditions and also in response to adverse circumstances, like drought stress. However, no study conducted to date has presented suitable reference genes, with stable expression along the day, for relative gene expression quantification in combined studies on drought stress and diurnal oscillations. Recently, water deficit responses have been associated with circadian clock oscillations at the transcription level, revealing the existence of hitherto unknown processes and increasing the demand for studies on plant responses to drought stress and its oscillation during the day. We performed data mining from a transcriptome-wide background using microarrays and RNA-seq databases to select an unpublished set of candidate reference genes, specifically chosen for the normalization of gene expression in studies on soybean under both drought stress and diurnal oscillations. Experimental validation and stability analysis in soybean plants submitted to drought stress and sampled during a 24 h timecourse showed that four of these newer reference genes (FYVE, NUDIX, Golgin-84 and CYST) indeed exhibited greater expression stability than the conventionally used housekeeping genes (ELF1-β and β-actin) under these conditions. We also demonstrated the effect of using reference candidate genes with different stability values to normalize the relative expression data from a drought-inducible soybean gene (DREB5) evaluated in different periods of the day.

  18. Determination of viable Salmonellae from potable and source water through PMA assisted qPCR.

    PubMed

    Singh, Gulshan; Vajpayee, Poornima; Bhatti, Saurabh; Ronnie, Nirmala; Shah, Nimish; McClure, Peter; Shanker, Rishi

    2013-07-01

    Resource constrained countries identified as endemic zones for pathogenicity of Salmonella bear an economic burden due to recurring expenditure on medical treatment. qPCR used for Salmonella detection could not discriminate between viable and nonviable cells. Propidium monoazide (PMA) that selectively penetrates nonviable cells to cross-link their DNA, was coupled with ttr gene specific qPCR for quantifying viable salmonellae in source/potable waters collected from a north Indian city. Source water (raw water for urban potable water supply) and urban potable water exhibited viable salmonellae in the range of 2.1×10(4)-2.6×10(6) and 2-7160CFU/100mL, respectively. Potable water at water works exhibited DNA from dead cells but no viable cells were detected. PMA assisted qPCR could specifically detect low numbers of live salmonellae in Source and potable waters. This strategy can be used in surveillance of urban potable water distribution networks to map contamination points for better microbial risk management.

  19. Quantification of Salmonella Typhi in water and sediments by molecular-beacon based qPCR.

    PubMed

    Rani, Neetika; Vajpayee, Poornima; Bhatti, Saurabh; Singh, Smriti; Shanker, Rishi; Gupta, Kailash Chand

    2014-10-01

    A molecular-beacon based qPCR assay targeting staG gene was designed for specific detection and quantification of S. Typhi and validated against water and sediment samples collected from the river Ganga, Yamuna and their confluence on two days during Mahakumbha mela 2012-2013 (a) 18 December, 2012: before six major religious holy dips (Makar Sankranti, Paush Poornima, Mauni Amavasya, Basant Panchami, Maghi Poornima and Mahashivratri) (b) 10 February, 2013: after the holy dip was taken by over 3,00,00,000 devotees led by ascetics of Hindu sects at Sangam on 'Mauni Amavasya' (the most auspicious day of ritualistic mass bathing). The assay could detect linearly lowest 1 genomic equivalent per qPCR and is highly sensitive and selective for S. Typhi detection in presence of non specific DNA from other bacterial strains including S. Paratyphi A and S. Typhimurium. It has been observed that water and sediment samples exhibit S. Typhi. The mass holy dip by devotees significantly affected the water and sediment quality by enhancing the number of S. Typhi in the study area. The qPCR developed in the study might be helpful in planning the intervention and prevention strategies for control of enteric fever outbreaks in endemic regions.

  20. Selection and Validation of Reference Genes for qRT-PCR Expression Analysis of Candidate Genes Involved in Olfactory Communication in the Butterfly Bicyclus anynana

    PubMed Central

    Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M.

    2015-01-01

    Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression

  1. Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

    PubMed

    Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M

    2015-01-01

    Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression

  2. Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.)

    PubMed Central

    2014-01-01

    Background Teak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak. Results Identification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), β-Tubulin (Β-Tub), ubiquitin (Ubq), elongation factor 1-α (Ef-1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1α are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1α as internal controls. These analyses exposed a consistent expression pattern with both reference genes. Conclusion This study

  3. A simple analytical and experimental procedure for selection of reference genes for reverse-transcription quantitative PCR normalization data.

    PubMed

    Manjarin, R; Trottier, N L; Weber, P S; Liesman, J S; Taylor, N P; Steibel, J P

    2011-10-01

    Variation in cellular activity in a tissue induces changes in RNA concentration, which affects the validity of gene mRNA abundance analyzed by reverse transcription quantitative PCR (RT-qPCR). A common way of accounting for such variation consists of the use of reference genes for normalization. Programs such as geNorm may be used to select suitable reference genes, although a large set of genes that are not co-regulated must be analyzed to obtain accurate results. The objective of this study was to propose an alternative experimental and analytical protocol to assess the invariance of reference genes in porcine mammary tissue using mammary RNA and DNA concentrations as correction factors. Mammary glands were biopsied from 4 sows on d 110 of gestation (prepartum), on d 5 (early) and 17 (peak) of lactation, and on d 5 after weaning (postweaning). Relative expression of 7 potential reference genes, API5, MRPL39, VAPB, ACTB, GAPDH, RPS23, and MTG1, and one candidate gene, SLC7A1, was quantified by RT-qPCR using a relative standard curve approach. Variation in gene expression levels, measured as cycles to threshold at each stage of mammary physiological activity, was tested using a linear mixed model fitting RNA and DNA concentrations as covariates. Results were compared with those obtained with geNorm analysis, and genes selected by each method were used to normalize SLC7A1. Quantified relative mRNA abundance of GAPDH and MRPL39 remained unchanged across stages of mammary physiological activity after accounting for changes in tissue RNA and DNA concentration. In contrast, geNorm analysis selected MTG1, MRPL39, and VAPB as the best reference genes. However, when target gene SLC7A1 was normalized with genes selected either based on our proposed protocol or by geNorm, fold changes in mRNA abundance did not differ. In conclusion, the proposed analytical protocol assesses expression invariance of potential reference genes by accounting for variation in tissue RNA and DNA

  4. Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop, Plukenetia volubilis, by Real-Time Quantitative PCR.

    PubMed

    Niu, Longjian; Tao, Yan-Bin; Chen, Mao-Sheng; Fu, Qiantang; Li, Chaoqiong; Dong, Yuling; Wang, Xiulan; He, Huiying; Xu, Zeng-Fu

    2015-06-03

    Real-time quantitative PCR (RT-qPCR) is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis), a promising oilseed crop known for its polyunsaturated fatty acid (PUFA)-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt), BestKeeper, geNorm, and NormFinder) were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13), cyclophilin (CYC) and elongation factor-1alpha (EF1α) were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII) were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi.

  5. Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop, Plukenetia volubilis, by Real-Time Quantitative PCR

    PubMed Central

    Niu, Longjian; Tao, Yan-Bin; Chen, Mao-Sheng; Fu, Qiantang; Li, Chaoqiong; Dong, Yuling; Wang, Xiulan; He, Huiying; Xu, Zeng-Fu

    2015-01-01

    Real-time quantitative PCR (RT-qPCR) is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis), a promising oilseed crop known for its polyunsaturated fatty acid (PUFA)-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt), BestKeeper, geNorm, and NormFinder) were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13), cyclophilin (CYC) and elongation factor-1alpha (EF1α) were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII) were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi. PMID:26047338

  6. RefPrimeCouch—a reference gene primer CouchApp

    PubMed Central

    Silbermann, Jascha; Wernicke, Catrin; Pospisil, Heike; Frohme, Marcus

    2013-01-01

    To support a quantitative real-time polymerase chain reaction standardization project, a new reference gene database application was required. The new database application was built with the explicit goal of simplifying not only the development process but also making the user interface more responsive and intuitive. To this end, CouchDB was used as the backend with a lightweight dynamic user interface implemented client-side as a one-page web application. Data entry and curation processes were streamlined using an OpenRefine-based workflow. The new RefPrimeCouch database application provides its data online under an Open Database License. Database URL: http://hpclife.th-wildau.de:5984/rpc/_design/rpc/view.html PMID:24368831

  7. Evaluation of reference genes for real-time PCR studies of Brazilian Somalis sheep infected by gastrointestinal nematodes

    PubMed Central

    2010-01-01

    Precise normalization with reference genes is necessary, in order to obtain reliable relative expression data in response to gastrointestinal nematode infection. By using sheep from temperate regions as models, three reference genes, viz., ribosomal protein LO (RPLO), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and succinate dehydrogenase complex subunit A (SDHA), were investigated in the abomasum, abomasal lymph nodes and small intestine of Brazilian Somalis sheep, either resistant or susceptible to gastrointestinal nematodes infections. Real time PCR was carried out by using SYBR Green I dye, and gene stability was tested by geNorm. RPLO was an ideal reference gene, since its expression was constant across treatments, presented lower variation, and was ranked as the most stable in abomasum and lymph node tissues. On the other hand, SDHA was the most stable in the small intestine followed by RPLO and GAPDH. These findings demonstrate the importance of correctly choosing reference genes prior to relative quantification. In addition, we determined that reference genes used in sheep from temperate regions, when properly tested, can be applied in animals from tropical regions such as the Brazilian Somalis sheep. PMID:21637421

  8. Certified DNA Reference Materials to Compare HER2 Gene Amplification Measurements Using Next-Generation Sequencing Methods.

    PubMed

    Lih, Chih-Jian; Si, Han; Das, Biswajit; Harrington, Robin D; Harper, Kneshay N; Sims, David J; McGregor, Paul M; Camalier, Corinne E; Kayserian, Andrew Y; Williams, P Mickey; He, Hua-Jun; Almeida, Jamie L; Lund, Steve P; Choquette, Steve; Cole, Kenneth D

    2016-09-01

    The National Institute of Standards and Technology (NIST) Standard Reference Materials 2373 is a set of genomic DNA samples prepared from five breast cancer cell lines with certified values for the ratio of the HER2 gene copy number to the copy numbers of reference genes determined by real-time quantitative PCR and digital PCR. Targeted-amplicon, whole-exome, and whole-genome sequencing measurements were used with the reference material to compare the performance of both the laboratory steps and the bioinformatic approaches of the different methods using a range of amplification ratios. Although good reproducibility was observed in each next-generation sequencing method, slightly different HER2 copy numbers associated with platform-specific biases were obtained. This study clearly demonstrates the value of Standard Reference Materials 2373 as reference material and as a calibrator for evaluating assay performance as well as for increasing confidence in reporting HER2 amplification for clinical applications.

  9. Selection of reference genes from two leafhopper species challenged by phytoplasma infection, for gene expression studies by RT-qPCR

    PubMed Central

    2013-01-01

    Background Phytoplasmas are phloem-limited phytopathogenic wall-less bacteria and represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. For gene expression studies based on mRNA quantification by RT-qPCR, stability of housekeeping genes is crucial. The aim of this study was the identification of reference genes to study the effect of phytoplasma infection on gene expression of two leafhopper vector species. The identified reference genes will be useful tools to investigate differential gene expression of leafhopper vectors upon phytoplasma infection. Results The expression profiles of ribosomal 18S, actin, ATP synthase β, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and tropomyosin were determined in two leafhopper vector species (Hemiptera: Cicadellidae), both healthy and infected by “Candidatus Phytoplasma asteris” (chrysanthemum yellows phytoplasma strain, CYP). Insects were analyzed at three different times post acquisition, and expression stabilities of the selected genes were evaluated with BestKeeper, geNorm and Normfinder algorithms. In Euscelidius variegatus, all genes under all treatments were stable and could serve as reference genes. In Macrosteles quadripunctulatus, BestKeeper and Normfinder analysis indicated ATP synthase β, tropomyosin and GAPDH as the most stable, whereas geNorm identified reliable genes only for early stages of infection. Conclusions In this study a validation of five candidate reference genes was performed with three algorithms, and housekeeping genes were identified for over time transcript profiling of two leafhopper vector species infected by CYP. This work set up an experimental system to study the molecular basis of phytoplasma multiplication in the insect body, in order to elucidate mechanisms of vector specificity. Most of the sequences provided in this study are new for leafhoppers, which are vectors of economically

  10. Identification of Reliable Reference Genes for Quantification of MicroRNAs in Serum Samples of Sulfur Mustard-Exposed Veterans

    PubMed Central

    Gharbi, Sedigheh; Shamsara, Mehdi; Khateri, Shahriar; Soroush, Mohammad Reza; Ghorbanmehr, Nassim; Tavallaei, Mahmood; Nourani, Mohammad Reza; Mowla, Seyed Javad

    2015-01-01

    Objective In spite of accumulating information about pathological aspects of sulfur mustard (SM), the precise mechanism responsible for its effects is not well understood. Circulating microRNAs (miRNAs) are promising biomarkers for disease diagnosis and prognosis. Accurate normalization using appropriate reference genes, is a critical step in miRNA expression studies. In this study, we aimed to identify appropriate reference gene for microRNA quantification in serum samples of SM victims. Materials and Methods In this case and control experimental study, using quantitative real-time polymerase chain reaction (qRT-PCR), we evaluated the suitability of a panel of small RNAs including SNORD38B, SNORD49A, U6, 5S rRNA, miR-423-3p, miR-191, miR-16 and miR-103 in sera of 28 SM-exposed veterans of Iran-Iraq war (1980-1988) and 15 matched control volunteers. Different statistical algorithms including geNorm, Normfinder, best-keeper and comparative delta-quantification cycle (Cq) method were employed to find the least variable reference gene. Results miR-423-3p was identified as the most stably expressed reference gene, and miR- 103 and miR-16 ranked after that. Conclusion We demonstrate that non-miRNA reference genes have the least stabil- ity in serum samples and that some house-keeping miRNAs may be used as more reliable reference genes for miRNAs in serum. In addition, using the geometric mean of two reference genes could increase the reliability of the normalizers. PMID:26464821

  11. Selection and assessment of reference genes for quantitative PCR normalization in migratory locust Locusta migratoria (Orthoptera: Acrididae).

    PubMed

    Yang, Qingpo; Li, Zhen; Cao, Jinjun; Zhang, Songdou; Zhang, Huaijiang; Wu, Xiaoyun; Zhang, Qingwen; Liu, Xiaoxia

    2014-01-01

    Locusta migratoria is a classic hemimetamorphosis insect and has caused widespread economic damage to crops as a migratory pest. Researches on the expression pattern of functional genes in L. migratoria have drawn focus in recent years, especially with the release of genome information. Real-time quantitative PCR is the most reproducible and sensitive approach for detecting transcript expression levels of target genes, but optimal internal standards are key factors for its accuracy and reliability. Therefore, it's necessary to provide a systematic stability assessment of internal control for well-performed tests of target gene expression profile. In this study, twelve candidate genes (Ach, Act, Cht2, EF1α, RPL32, Hsp70, Tub, RP49, SDH, GAPDH, 18S, and His) were analyzed with four statistical methods: the delta Ct approach, geNorm, Bestkeeper and NormFinder. The results from these analyses aimed to choose the best suitable reference gene across different experimental situations for gene profile study in L. migratoria. The result demonstrated that for different developmental stages, EF1α, Hsp70 and RPL32 exhibited the most stable expression status for all samples; EF1α and RPL32 were selected as the best reference genes for studies involving embryo and larvae stages, while SDH and RP49 were identified for adult stage. The best-ranked reference genes across different tissues are RPL32, Hsp70 and RP49. For abiotic treatments, the most appropriate genes we identified were as follows: Act and SDH for larvae subjected to different insecticides; RPL32 and Ach for larvae exposed to different temperature treatments; and Act and Ach for larvae suffering from starvation. The present report should facilitate future researches on gene expression in L. migratoria with accessibly optimal reference genes under different experimental contexts.

  12. Evaluation and Selection of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Withania somnifera (L.) Dunal

    PubMed Central

    Singh, Varinder; Kaul, Sunil C.; Wadhwa, Renu; Pati, Pratap Kumar

    2015-01-01

    Quantitative real-time PCR (qRT-PCR) is now globally used for accurate analysis of transcripts levels in plants. For reliable quantification of transcripts, identification of the best reference genes is a prerequisite in qRT-PCR analysis. Recently, Withania somnifera has attracted lot of attention due to its immense therapeutic potential. At present, biotechnological intervention for the improvement of this plant is being seriously pursued. In this background, it is important to have comprehensive studies on finding suitable reference genes for this high valued medicinal plant. In the present study, 11 candidate genes were evaluated for their expression stability under biotic (fungal disease), abiotic (wounding, salt, drought, heat and cold) stresses, in different plant tissues and in response to various plant growth regulators (methyl jasmonate, salicylic acid, abscisic acid). The data as analyzed by various software packages (geNorm, NormFinder, Bestkeeper and ΔCt method) suggested that cyclophilin (CYP) is a most stable gene under wounding, heat, methyl jasmonate, different tissues and all stress conditions. T-SAND was found to be a best reference gene for salt and salicylic acid (SA) treated samples, while 26S ribosomal RNA (26S), ubiquitin (UBQ) and beta-tubulin (TUB) were the most stably expressed genes under drought, biotic and cold treatment respectively. For abscisic acid (ABA) treated samples 18S-rRNA was found to stably expressed gene. Finally, the relative expression level of the three genes involved in the withanolide biosynthetic pathway was detected to validate the selection of reliable reference genes. The present work will significantly contribute to gene analysis studies in W. somnifera and facilitate in improving the quality of gene expression data in this plant as well as and other related plant species. PMID:25769035

  13. Identification and validation of reference genes for quantification of target gene expression with quantitative real-time PCR for tall fescue under four abiotic stresses.

    PubMed

    Yang, Zhimin; Chen, Yu; Hu, Baoyun; Tan, Zhiqun; Huang, Bingru

    2015-01-01

    Tall fescue (Festuca arundinacea Schreb.) is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41) was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.

  14. Identification and Validation of Reference Genes for Quantification of Target Gene Expression with Quantitative Real-time PCR for Tall Fescue under Four Abiotic Stresses

    PubMed Central

    Hu, Baoyun; Tan, Zhiqun; Huang, Bingru

    2015-01-01

    Tall fescue (Festuca arundinacea Schreb.) is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41) was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species. PMID:25786207

  15. Finding the joker among the maize endogenous reference genes for genetically modified organism (GMO) detection.

    PubMed

    Paternò, Annalisa; Marchesi, Ugo; Gatto, Francesco; Verginelli, Daniela; Quarchioni, Cinzia; Fusco, Cristiana; Zepparoni, Alessia; Amaddeo, Demetrio; Ciabatti, Ilaria

    2009-12-09

    The comparison of five real-time polymerase chain reaction (PCR) methods targeted at maize ( Zea mays ) endogenous sequences is reported. PCR targets were the alcohol dehydrogenase (adh) gene for three methods and high-mobility group (hmg) gene for the other two. The five real-time PCR methods have been checked under repeatability conditions at several dilution levels on both pooled DNA template from several genetically modified (GM) maize certified reference materials (CRMs) and single CRM DNA extracts. Slopes and R(2) coefficients of all of the curves obtained from the adopted regression model were compared within the same method and among all of the five methods, and the limit of detection and limit of quantitation were analyzed for each PCR system. Furthermore, method equivalency was evaluated on the basis of the ability to estimate the target haploid genome copy number at each concentration level. Results indicated that, among the five methods tested, one of the hmg-targeted PCR systems can be considered equivalent to the others but shows the best regression parameters and a higher repeteability along the dilution range. Thereby, it is proposed as a valid module to be coupled to different event-specific real-time PCR for maize genetically modified organism (GMO) quantitation. The resulting practicability improvement on the analytical control of GMOs is discussed.

  16. Is qPCR a Reliable Indicator of Cyanotoxin Risk in Freshwater?

    PubMed Central

    Pacheco, Ana Beatriz F.; Guedes, Iame A.; Azevedo, Sandra M.F.O.

    2016-01-01

    The wide distribution of cyanobacteria in aquatic environments leads to the risk of water contamination by cyanotoxins, which generate environmental and public health issues. Measurements of cell densities or pigment contents allow both the early detection of cellular growth and bloom monitoring, but these methods are not sufficiently accurate to predict actual cyanobacterial risk. To quantify cyanotoxins, analytical methods are considered the gold standards, but they are laborious, expensive, time-consuming and available in a limited number of laboratories. In cyanobacterial species with toxic potential, cyanotoxin production is restricted to some strains, and blooms can contain varying proportions of both toxic and non-toxic cells, which are morphologically indistinguishable. The sequencing of cyanobacterial genomes led to the description of gene clusters responsible for cyanotoxin production, which paved the way for the use of these genes as targets for PCR and then quantitative PCR (qPCR). Thus, the quantification of cyanotoxin genes appeared as a new method for estimating the potential toxicity of blooms. This raises a question concerning whether qPCR-based methods would be a reliable indicator of toxin concentration in the environment. Here, we review studies that report the parallel detection of microcystin genes and microcystin concentrations in natural populations and also a smaller number of studies dedicated to cylindrospermopsin and saxitoxin. We discuss the possible issues associated with the contradictory findings reported to date, present methodological limitations and consider the use of qPCR as an indicator of cyanotoxin risk. PMID:27338471

  17. Genome-Wide Identification and Validation of Reference Genes in Infected Tomato Leaves for Quantitative RT-PCR Analyses

    PubMed Central

    Müller, Oliver A.; Grau, Jan; Thieme, Sabine; Prochaska, Heike; Adlung, Norman; Sorgatz, Anika; Bonas, Ulla

    2015-01-01

    The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv) causes bacterial spot disease of pepper and tomato by direct translocation of type III effector proteins into the plant cell cytosol. Once in the plant cell the effectors interfere with host cell processes and manipulate the plant transcriptome. Quantitative RT-PCR (qRT-PCR) is usually the method of choice to analyze transcriptional changes of selected plant genes. Reliable results depend, however, on measuring stably expressed reference genes that serve as internal normalization controls. We identified the most stably expressed tomato genes based on microarray analyses of Xcv-infected tomato leaves and evaluated the reliability of 11 genes for qRT-PCR studies in comparison to four traditionally employed reference genes. Three different statistical algorithms, geNorm, NormFinder and BestKeeper, concordantly determined the superiority of the newly identified reference genes. The most suitable reference genes encode proteins with homology to PHD finger family proteins and the U6 snRNA-associated protein LSm7. In addition, we identified pepper orthologs and validated several genes as reliable normalization controls for qRT-PCR analysis of Xcv-infected pepper plants. The newly identified reference genes will be beneficial for future qRT-PCR studies of the Xcv-tomato and Xcv-pepper pathosystems, as well as for the identification of suitable normalization controls for qRT-PCR studies of other plant-pathogen interactions, especially, if related plant species are used in combination with bacterial pathogens. PMID:26313760

  18. Genome-Wide Identification and Validation of Reference Genes in Infected Tomato Leaves for Quantitative RT-PCR Analyses.

    PubMed

    Müller, Oliver A; Grau, Jan; Thieme, Sabine; Prochaska, Heike; Adlung, Norman; Sorgatz, Anika; Bonas, Ulla

    2015-01-01

    The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv) causes bacterial spot disease of pepper and tomato by direct translocation of type III effector proteins into the plant cell cytosol. Once in the plant cell the effectors interfere with host cell processes and manipulate the plant transcriptome. Quantitative RT-PCR (qRT-PCR) is usually the method of choice to analyze transcriptional changes of selected plant genes. Reliable results depend, however, on measuring stably expressed reference genes that serve as internal normalization controls. We identified the most stably expressed tomato genes based on microarray analyses of Xcv-infected tomato leaves and evaluated the reliability of 11 genes for qRT-PCR studies in comparison to four traditionally employed reference genes. Three different statistical algorithms, geNorm, NormFinder and BestKeeper, concordantly determined the superiority of the newly identified reference genes. The most suitable reference genes encode proteins with homology to PHD finger family proteins and the U6 snRNA-associated protein LSm7. In addition, we identified pepper orthologs and validated several genes as reliable normalization controls for qRT-PCR analysis of Xcv-infected pepper plants. The newly identified reference genes will be beneficial for future qRT-PCR studies of the Xcv-tomato and Xcv-pepper pathosystems, as well as for the identification of suitable normalization controls for qRT-PCR studies of other plant-pathogen interactions, especially, if related plant species are used in combination with bacterial pathogens.

  19. An alternative and sensitive method based on LCM and Q-PCR for HER2 testing in breast cancer.

    PubMed

    Fetica, Bogdan; Balacescu, Ovidiu; Balacescu, Loredana; Rus, Meda; Berindan-Neagoe, Ioana

    2014-01-01

    Nowadays, HER2 testing in breast cancer represents a necessity for both prognostic and therapy. Despite widespread use of immunohistochemistry (IHC) for assessing HER2 status, there are some limitations to identify truly negative or positive HER2 cases. Fluorescence in situ hybridization (FISH) or chromogenic in situ hybridization (CISH) could solve the equivocal HER2 IHC cases but there is no consensus on which is the best method. Consequently, finding a sensitive method for HER2 testing is critical for the management of the disease. In addition, tumor heterogeneity is an important factor which could affect accuracy of molecular diagnostics. Laser capture micro-dissection (LCM) is used to isolate pure cell populations from heterogeneous tumor tissue. The combination between LCM and quantitative polymerase chain reaction (Q-PCR), the gold standard in molecular biology for quantifying gene amplification levels, could define an important tool to improve the molecular diagnostics of HER2 status.In our pilot study we used LCM and Q-PCR to evaluate HER2 gene amplification for invasive breast carcinoma samples. The samples were selected based on HER2 status assessed by IHC and CISH. Our results demonstrated high sensitivity of Q-PCR for assessing HER2 DNA amplification as well as a good concordance between Q-PCR and IHC/ CISH assay.

  20. Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus).

    PubMed

    Yang, Chang Geng; Wang, Xian Li; Tian, Juan; Liu, Wei; Wu, Fan; Jiang, Ming; Wen, Hua

    2013-09-15

    Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) has been used frequently to study gene expression related to fish immunology. In such studies, a stable reference gene should be selected to correct the expression of the target gene. In this study, seven candidate reference genes (glyceraldehyde-3-phosphate dehydrogenase (GADPH), ubiquitin-conjugating enzyme (UBCE), 18S ribosomal RNA (18S rRNA), beta-2-microglobulin (B2M), elongation factor 1 alpha (EF1A), tubulin alpha chain-like (TUBA) and beta actin (ACTB)), were selected to analyze their stability and normalization in seven tissues (liver, spleen, kidney, brain, heart, muscle and intestine) of Nile tilapia (Oreochromis niloticus) challenged with Streptococcus agalactiae or Streptococcus iniae, respectively. The results showed that all the candidate reference genes exhibited tissue-dependent transcriptional variations. With PBS injection as a control, UBCE was the most stable and suitable single reference gene in the intestine, liver, brain, kidney, and spleen after S. iniae infection, and in the liver, kidney, and spleen after S. agalactiae infection. EF1A was the most suitable in heart and muscle after S. iniae or S. agalactiae infection. GADPH was the most suitable gene in intestine and brain after S. agalactiae infection. In normal conditions, UBCE and 18S rRNA were the most stably expressed genes across the various tissues. These results showed that for RT-qPCR analysis of tilapia, selecting two or more reference genes may be more suitable for cross-tissue analysis of gene expression.

  1. Selection and Evaluation of Potential Reference Genes for Gene Expression Analysis in the Brown Planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) Using Reverse-Transcription Quantitative PCR

    PubMed Central

    Zhu, Xun; Wan, Hu; Shakeel, Muhammad; Zhan, Sha; Jin, Byung-Rae; Li, Jianhong

    2014-01-01

    The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for

  2. Selection of Suitable Reference Genes for Analysis of Salivary Transcriptome in Non-Syndromic Autistic Male Children

    PubMed Central

    Panahi, Yasin; Salasar Moghaddam, Fahimeh; Ghasemi, Zahra; Hadi Jafari, Mandana; Shervin Badv, Reza; Eskandari, Mohamad Reza; Pedram, Mehrdad

    2016-01-01

    Childhood autism is a severe form of complex genetically heterogeneous and behaviorally defined set of neurodevelopmental diseases, collectively termed as autism spectrum disorders (ASD). Reverse transcriptase quantitative real-time PCR (RT-qPCR) is a highly sensitive technique for transcriptome analysis, and it has been frequently used in ASD gene expression studies. However, normalization to stably expressed reference gene(s) is necessary to validate any alteration reported at the mRNA level for target genes. The main goal of the present study was to find the most stable reference genes in the salivary transcriptome for RT-qPCR analysis in non-syndromic male childhood autism. Saliva samples were obtained from nine drug naïve non-syndromic male children with autism and also sex-, age-, and location-matched healthy controls using the RNA-stabilizer kit from DNA Genotek. A systematic two-phased measurement of whole saliva mRNA levels for eight common housekeeping genes (HKGs) was carried out by RT-qPCR, and the stability of expression for each candidate gene was analyzed using two specialized algorithms, geNorm and NormFinder, in parallel. Our analysis shows that while the frequently used HKG ACTB is not a suitable reference gene, the combination of GAPDH and YWHAZ could be recommended for normalization of RT-qPCR analysis of salivary transcriptome in non-syndromic autistic male children. PMID:27754318

  3. Selection of Suitable Reference Genes for Analysis of Salivary Transcriptome in Non-Syndromic Autistic Male Children.

    PubMed

    Panahi, Yasin; Salasar Moghaddam, Fahimeh; Ghasemi, Zahra; Hadi Jafari, Mandana; Shervin Badv, Reza; Eskandari, Mohamad Reza; Pedram, Mehrdad

    2016-10-12

    Childhood autism is a severe form of complex genetically heterogeneous and behaviorally defined set of neurodevelopmental diseases, collectively termed as autism spectrum disorders (ASD). Reverse transcriptase quantitative real-time PCR (RT-qPCR) is a highly sensitive technique for transcriptome analysis, and it has been frequently used in ASD gene expression studies. However, normalization to stably expressed reference gene(s) is necessary to validate any alteration reported at the mRNA level for target genes. The main goal of the present study was to find the most stable reference genes in the salivary transcriptome for RT-qPCR analysis in non-syndromic male childhood autism. Saliva samples were obtained from nine drug naïve non-syndromic male children with autism and also sex-, age-, and location-matched healthy controls using the RNA-stabilizer kit from DNA Genotek. A systematic two-phased measurement of whole saliva mRNA levels for eight common housekeeping genes (HKGs) was carried out by RT-qPCR, and the stability of expression for each candidate gene was analyzed using two specialized algorithms, geNorm and NormFinder, in parallel. Our analysis shows that while the frequently used HKG ACTB is not a suitable reference gene, the combination of GAPDH and YWHAZ could be recommended for normalization of RT-qPCR analysis of salivary transcriptome in non-syndromic autistic male children.

  4. Design and validation of a qPCR assay for accurate detection and initial serogrouping of Legionella pneumophila in clinical specimens by the ESCMID Study Group for Legionella Infections (ESGLI).

    PubMed

    Mentasti, M; Kese, D; Echahidi, F; Uldum, S A; Afshar, B; David, S; Mrazek, J; De Mendonça, R; Harrison, T G; Chalker, V J

    2015-07-01

    Prompt detection of Legionella pneumophila is essential for rapid investigation of legionellosis. Furthermore, as the majority of L. pneumophila infections are caused by serogroup 1 (sg1) strains, rapid identification of such strains can be critical in both routine and outbreak scenarios. The ESCMID Study Group for Legionella Infections (ESGLI) was established in 2012 and immediately identified as a priority the validation of a reliable, easy to perform and interpret, cost-effective qPCR assay to standardise the detection of L. pneumophila DNA amongst members. A novel L. pneumophila assay targeting the mip gene was designed and combined with previously published methodologies amplifying the sg1 marker (wzm) and the green fluorescent protein gene (gfp) internal process control. The resulting triplex assay was validated internationally on the three qPCR platforms used by the majority of European Legionella reference laboratories: ABI 7500 (Life Technologies), LightCycler 480 Instrument II (Roche) and Rotor-Gene Q (Qiagen). Clinical and EQA specimens were tested together with a large panel of strains (251 in total) to validate the assay. The assay proved to be 100% specific for L. pneumophila and sg1 DNA both in silico and in vitro. Efficiency values for mip and wzm assays ranged between 91.97 and 97.69%. Limit of detection values estimated with 95% confidence were adopted for mip and wzm assays on all three qPCR platforms. Inhibition was not observed. This study describes a robust assay that could be widely implemented to standardise the molecular detection of L. pneumophila among ESGLI laboratories and beyond.

  5. qPCR analysis of carbon, nitrogen, and arsenic cycling in Zetaproteobacteria-dominated microbial mats

    NASA Astrophysics Data System (ADS)

    Jesser, K. J.; Fullerton, H.; Hilton, T. S.; Kimber, J.; Hager, K.; Moyer, C. L.

    2013-12-01

    The recently discovered Zetaproteobacteria represent a novel class of Proteobacteria which oxidize Fe(II) to Fe(III) to fix CO2 at hydrothermal vents. Zetaproteobacteria were first discovered at Lo'ihi Seamount, located 35 km southeast of the big island of Hawai'i and characterized by low-temperature diffuse hydrothermal vents. The hydrothermal vents at Lo'ihi are surrounded by luxuriant iron-rich microbial mats dominated by Zetaproteobacteria. We aim to use real-time quantitative PCR (qPCR) to quantify functional genes associated with the microbial carbon, nitrogen, and arsenic cycles in complex Zetaproteobacteria- dominated iron mat communities. Unique qPCR primer sets have been developed based on Illumina next-generation sequence data from an iron mat collected in 2009 at Lo'ihi. These primers target the sequences for arsenate reductase and nitrite reductase, genes associated with arsenic detoxification and denitrification, respectively. Additionally, we are utilizing published primer sets to quantify genes associated with autotrophic carbon and nitrogen fixation pathways. Genomic DNA was isolated from microbial mats at multiple vent sites with varying temperatures and fluid flow during our 2013 expedition to Lo'ihi. The qPCR data for these samples can be used to draw correlations among fine scale mat structures and nutrient cycling processes across diverse mat morphologies, as previous research has identified unique microbial communities and metabolic strategies associated with distinct mat morphologies. This work will enable us to better identify samples for further molecular analysis, and may provide insights into the evolutionary history and metabolic functionality of various mat morphotypes. We hypothesize that Zetaproteobacteria act as ecosystem engineers, driving the structure and function of iron mat ecosystems.

  6. Identification of Appropriate Reference Genes for Normalization of miRNA Expression in Grafted Watermelon Plants under Different Nutrient Stresses

    PubMed Central

    Wu, Weifang; Deng, Qin; Shi, Pibiao; Yang, Jinghua; Hu, Zhongyuan; Zhang, Mingfang

    2016-01-01

    Watermelon (Citrullus lanatus) is a globally important crop belonging to the family Cucurbitaceae. The grafting technique is commonly used to improve its tolerance to stress, as well as to enhance its nutrient uptake and utilization. It is believed that miRNA is most likely involved in its nutrient-starvation response as a graft-transportable signal. The quantitative real-time reverse transcriptase polymerase chain reaction is the preferred method for miRNA functional analysis, in which reliable reference genes for normalization are crucial to ensure the accuracy. The purpose of this study was to select appropriate reference genes in scion (watermelon) and rootstocks (squash and bottle gourd) of grafted watermelon plants under normal growth conditions and nutrient stresses (nitrogen and phosphorus starvation). Under nutrient starvation, geNorm identified miR167c and miR167f as two most stable genes in both watermelon leaves and squash roots. miR166b was recommended by both geNorm and NormFinder as the best reference in bottle gourd roots under nutrient limitation. Expression of a new Cucurbitaceae miRNA, miR85, was used to validate the reliability of candidate reference genes under nutrient starvation. Moreover, by comparing several target genes expression in qRT-PCR analysis with those in RNA-seq data, miR166b and miR167c were proved to be the most suitable reference genes to normalize miRNA expression under normal growth condition in scion and rootstock tissues, respectively. This study represents the first comprehensive survey of the stability of miRNA reference genes in Cucurbitaceae and provides valuable information for investigating more accurate miRNA expression involving grafted watermelon plants. PMID:27749935

  7. Novel and sensitive qPCR assays for the detection and identification of aspergillosis causing species.

    PubMed

    Paholcsek, Melinda; Leiter, Eva; Markovics, Arnold; Biró, Sándor

    2014-09-01

    Despite concerted efforts, diagnosis of aspergillosis is still a great challenge to clinical microbiology laboratories. Along with the requirement for high sensitivity and specificity, species-specific identification is important. We developed rapid, sensitive and species-specific qPCR assays using the TaqMan technology for the detection and identification of Aspergillus fumigatus and Aspergillus terreus. The assays were designed to target orthologs of the Streptomyces factor C gene that are only found in a few species of filamentous fungi. Fungi acquired this gene through horizontal gene transfer and divergence of the gene allows identification of species. The assays have potential as a molecular diagnosis tool for the early detection of fungal infection caused by Aspergillus fumigatus and Aspergillus terreus, which merits future diagnostic studies. The assays were sensitive enough to detect a few genomic equivalents in blood samples.

  8. Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

    PubMed Central

    Chan, Pek-Lan; Rose, Ray J.; Abdul Murad, Abdul Munir; Zainal, Zamri; Leslie Low, Eng-Ti; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

    2014-01-01

    Background The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. Results In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Conclusions Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate

  9. Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation.

    PubMed

    Zhang, Juan; Tang, Hongju; Zhang, Yuqing; Deng, Ruyuan; Shao, Li; Liu, Yun; Li, Fengying; Wang, Xiao; Zhou, Libin

    2014-05-01

    Quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important in the effort to gain insight into the molecular mechanisms underlying adipogenesis. However, the expression profile of a target gene may be misinterpreted due to the unstable expression of the reference genes under different experimental conditions. Therefore, in this study, we investigated the expression stability of 10 commonly used reference genes during 3T3-L1 adipocyte differentiation. The mRNA expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transferrin receptor (TFRC) significantly increased during the course of 3T3-L1 adipocyte differentiation, which was decreased by berberine, an inhibitor of adipogenesis. Three popular algorithms, GeNorm, NormFinder and BestKeeper, identified 18 ribosomal RNA and hydroxymethylbilane synthase (HMBS) as the most stable reference genes, while GAPDH and TFRC were the least stable ones. Peptidylprolyl isomerase A [PIPA (cyclophilin A)], ribosomal protein, large, P0 (36-B4), beta-2-microglobulin (B2M), α1-tubulin, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and β-actin showed relatively stable expression levels. The choice of reference genes with various expression stabilities exerted a profound influence on the expression profiles of 2 target genes, peroxisome proliferator-activated receptor (PPAR)γ2 and C/EBPα. In addition, western blot analysis revealed that the increased protein expression of GAPDH was markedly inhibited by berberine during adipocyte differentiation. This study highlights the importance of selecting suitable reference genes for qRT-PCR studies of gene expression during the process of adipogenesis.

  10. Spatial and temporal consistency of putative reference genes for real-time PCR in a model tapeworm.

    PubMed

    Pouchkina-Stantcheva, Natalia N; Cunningham, Lucas J; Olson, Peter D

    2011-12-01

    Relative quantification of gene expression by real-time PCR relies on the use of reference genes whose expressed levels remain consistent across experimental conditions. Here we compare expression levels of commonly employed endogenous housekeeping genes against a developmental regulatory gene in the model tapeworm Hymenolepis microstoma, examining variation both spatially across regions of the adult worm and temporally through the course of larval metamorphosis. β-Tubulin, RNA polymerase II and 60S ribosomal subunit L28 showed the most variance among candidate reference genes when comparing changes in expression along the anteroposterior gradient of development represented by the adult body, whereas expression of 18S rDNA and cyclic AMP were highly consistent and could be used reliably for relative quantification. The transcription factor Hox4, referenced to either 18S or cAMP, showed 3-fold higher expression levels in the neck region than in more mature regions of the strobila. In contrast, variance among samples representing progressive stages of larval metamorphosis were greater for candidate reference genes than for Hox4, indicating that none of the candidates can be used reliably for quantifying relative changes in gene expression during metamorphosis.

  11. Characterization of 137 Genomic DNA Reference Materials for 28 Pharmacogenetic Genes

    PubMed Central

    Pratt, Victoria M.; Everts, Robin E.; Aggarwal, Praful; Beyer, Brittany N.; Broeckel, Ulrich; Epstein-Baak, Ruth; Hujsak, Paul; Kornreich, Ruth; Liao, Jun; Lorier, Rachel; Scott, Stuart A.; Smith, Chingying Huang; Toji, Lorraine H.; Turner, Amy; Kalman, Lisa V.

    2017-01-01

    Pharmacogenetic testing is increasingly available from clinical laboratories. However, only a limited number of quality control and other reference materials are currently available to support clinical testing. To address this need, the Centers for Disease Control and Prevention–based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, has characterized 137 genomic DNA samples for 28 genes commonly genotyped by pharmacogenetic testing assays (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP4F2, DPYD, GSTM1, GSTP1, GSTT1, NAT1, NAT2, SLC15A2, SLC22A2, SLCO1B1, SLCO2B1, TPMT, UGT1A1, UGT2B7, UGT2B15, UGT2B17, and VKORC1). One hundred thirty-seven Coriell cell lines were selected based on ethnic diversity and partial genotype characterization from earlier testing. DNA samples were coded and distributed to volunteer testing laboratories for targeted genotyping using a number of commercially available and laboratory developed tests. Through consensus verification, we confirmed the presence of at least 108 variant pharmacogenetic alleles. These samples are also being characterized by other pharmacogenetic assays, including next-generation sequencing, which will be reported separately. Genotyping results were consistent among laboratories, with most differences in allele assignments attributed to assay design and variability in reported allele nomenclature, particularly for CYP2D6, UGT1A1, and VKORC1. These publicly available samples will help ensure the accuracy of pharmacogenetic testing. PMID:26621101

  12. Selection and evaluation of novel reference genes for quantitative reverse transcription PCR (qRT-PCR) based on genome and transcriptome data in Brassica napus L.

    PubMed

    Yang, Hongli; Liu, Jing; Huang, Shunmou; Guo, Tingting; Deng, Linbin; Hua, Wei

    2014-03-15

    Selection of reference genes in Brassica napus, a tetraploid (4×) species, is a very difficult task without information on genome and transcriptome. By now, only several traditional reference genes which show significant expression differentiation under different conditions are used in B. napus. In the present study, based on genome and transcriptome data of the rapeseed Zhongshuang-11 cultivar, 14 candidate reference genes were screened for investigation in different tissues, cultivars, and treated conditions of B. napus. These genes were as follows: ELF5, ENTH, F-BOX7, F-BOX2, FYPP1, GDI1, GYF, MCP2d, OTP80, PPR, SPOC, Unknown1, Unknown2 and UBA. Among them, excluding GYF and FYPP1, another 12 genes, were identified to perform better than traditional reference genes ACTIN7 and GAPDH. To further validate the accuracy of the newly developed reference genes in normalization, expression levels of BnCAT1 (B. napus catalase 1) in different rapeseed tissues and seedlings under stress conditions were normalized by the three most stable reference genes PPR, GDI1, and ENTH and little difference existed in normalization results. To the best of our knowledge, this is the first time B. napus reference genes have been provided with the help of complete genome and transcriptome information. The new reference genes provided in this study are more accurate than previously reported reference genes in quantifying expression levels of B. napus genes.

  13. Construction and application of a Korean reference panel for imputing classical alleles and amino acids of human leukocyte antigen genes.

    PubMed

    Kim, Kwangwoo; Bang, So-Young; Lee, Hye-Soon; Bae, Sang-Cheol

    2014-01-01

    Genetic variations of human leukocyte antigen (HLA) genes within the major histocompatibility complex (MHC) locus are strongly associated with disease susceptibility and prognosis for many diseases, including many autoimmune diseases. In this study, we developed a Korean HLA reference panel for imputing classical alleles and amino acid residues of several HLA genes. An HLA reference panel has potential for use in identifying and fine-mapping disease associations with the MHC locus in East Asian populations, including Koreans. A total of 413 unrelated Korean subjects were analyzed for single nucleotide polymorphisms (SNPs) at the MHC locus and six HLA genes, including HLA-A, -B, -C, -DRB1, -DPB1, and -DQB1. The HLA reference panel was constructed by phasing the 5,858 MHC SNPs, 233 classical HLA alleles, and 1,387 amino acid residue markers from 1,025 amino acid positions as binary variables. The imputation accuracy of the HLA reference panel was assessed by measuring concordance rates between imputed and genotyped alleles of the HLA genes from a subset of the study subjects and East Asian HapMap individuals. Average concordance rates were 95.6% and 91.1% at 2-digit and 4-digit allele resolutions, respectively. The imputation accuracy was minimally affected by SNP density of a test dataset for imputation. In conclusion, the Korean HLA reference panel we developed was highly suitable for imputing HLA alleles and amino acids from MHC SNPs in East Asians, including Koreans.

  14. Identification of Reference Genes for Quantitative Real Time PCR Assays in Aortic Tissue of Syrian Hamsters with Bicuspid Aortic Valve

    PubMed Central

    Rueda-Martínez, Carmen; Fernández, M. Carmen; Soto-Navarrete, María Teresa; Jiménez-Navarro, Manuel; Durán, Ana Carmen; Fernández, Borja

    2016-01-01

    Bicuspid aortic valve (BAV) is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40%) incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans. In this report, we evaluate the expression of ten candidate reference genes in aortic tissue of hamsters in order to identify housekeeping genes for normalization using quantitative real time PCR (RT-qPCR) assays. A total of 51 adult (180–240 days old) and 56 old (300–440 days old) animals were used. They belonged to a control strain of hamsters with normal, tricuspid aortic valve (TAV; n = 30), or to the affected strain of hamsters with TAV (n = 45) or BAV (n = 32). The expression stability of the candidate reference genes was determined by RT-qPCR using three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable reference genes for the three algorithms employed were Cdkn1β, G3pdh and Polr2a. We propose the use of Cdkn1β, or both Cdkn1β and G3pdh as reference genes for mRNA expression analyses in Syrian hamster aorta. PMID:27711171

  15. Reference gene selection for real-time quantitative PCR analysis of the mouse uterus in the peri-implantation period.

    PubMed

    Lin, Pengfei; Lan, Xiangli; Chen, Fenglei; Yang, Yanzhou; Jin, Yaping; Wang, Aihua

    2013-01-01

    The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR) is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several mouse models, including an early pregnancy, a pseudopregnancy, a delayed implantation and activation, an artificial decidualization and a hormonal treatment model; ten candidate reference genes (PPIA, RPLP0, HPRT1, GAPDH, ACTB, TBP, B2M, 18S, UBC and TUBA) that are found in uterine tissues were assessed for their suitability as internal controls for relative qRT-PCR quantification. GeNorm(PLUS), NormFinder, and BestKeeper were used to evaluate these candidate reference genes, and all of these methods identified RPLP0 and GAPDH as the most stable candidates and B2M and 18S as the least stable candidates. However, when the different models were analyzed separately, the reference genes exhibited some variation in their expression levels.

  16. Evaluation of Reference Genes for the Relative Quantification of Apple stem grooving virus and Apple mosaic virus in Apple Trees.

    PubMed

    Gadiou, S; Kundu, J K

    2012-06-01

    A SYBR Green(®)-based one step RT-qPCR assay was developed for the detection and quantification of Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV). The RT-qPCR assay employed seven plant-expressed genes-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA, ubiquitin, ribosomal protein S19, Rubisco, RNA polymerase subunit II and β-actin-as internal reference housekeeping genes in a relative quantification system in three apple cultivars (i.e. Idared, Champion, Fragrance). The average expression stability (M) found by GeNorm software suggest that GAPDH and S19 were the most stable reference genes. We propose employing GAPDH and S19 as housekeeping genes for accurate quantification of ASGV and ApMV in apple leaf samples. The detection limit for both viruses was found around 70 copies of viral genome by one-step RT-qPCR.

  17. Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies

    PubMed Central

    Spinsanti, Giacomo; Panti, Cristina; Lazzeri, Elisa; Marsili, Letizia; Casini, Silvia; Frati, Francesco; Fossi, Cristina Maria

    2006-01-01

    Background Odontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR) has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers. Results Ten commonly used housekeeping genes (HKGs) were partially sequenced in the striped dolphin (Stenella coeruleoalba) and, for each gene, PCR primer pairs were specifically designed and tested in qRT-PCR assays. The expression of these potential control genes was examined in 30 striped dolphin skin biopsy samples, obtained from specimens sampled in the north-western Mediterranean Sea. The stability of selected control genes was determined using three different specific VBA applets (geNorm, NormFinder and BestKeeper) which produce highly comparable results. Glyceraldehyde-3P-dehydrogenase (GAPDH) and tyrosine 3-monooxygenase (YWHAZ) always rank as the two most stably expressed HKGs according to the analysis with geNorm and Normfinder, and are defined as optimal control genes by BestKepeer. Ribosomal protein L4 (RPL4) and S18 (RPS18) also exhibit a remarkable stability of their expression levels. On the other hand, transferrin receptor (TFRC), phosphoglycerate kinase 1 (PGK1), hypoxanthine ribosyltransferase (HPRT1) and β-2-microglobin (B2M) show variable expression among the studied

  18. A qPCR method to characterize the sex type of the cell strains from rats.

    PubMed

    Xiang, Junbei; Li, Zhilin; Wan, Qian; Chen, Qiang; Liu, Mianxue; Jiang, Xiaohui; Xie, Linfeng

    2016-10-01

    A simple and fast method was established to identify the sex types of the rat-derived cell strains. The single copy X-chromosome-linked gene AR and the single copy Y-chromosome-linked gene Sry were both detected with qPCR for the rat genomic DNA sample and the AR/Sry ratio was calculated. According to the law of the AR/Sry ratio, a new method to identify the sex types of the rat-derived cell strains was developed. The new assay was proved effective. The new assay showed advantages over the traditional sex type identification PCR methods, which detected only the Sry gene. Moreover, the new method was used to identify the sex types of two rat-derived cell strains unknown for the sex types and the results were confirmed with the in situ hybridization. Finally, the problem of the cross contamination between the female and the male samples was addressed and discussed extensively.

  19. Exploiting a Reference Genome in Terms of Duplications: The Network of Paralogs and Single Copy Genes in Arabidopsis thaliana.

    PubMed

    Sangiovanni, Mara; Vigilante, Alessandra; Chiusano, Maria Luisa

    2013-12-09

    Arabidopsis thaliana became the model organism for plant studies because of its small diploid genome, rapid lifecycle and short adult size. Its genome was the first among plants to be sequenced, becoming the reference in plant genomics. However, the Arabidopsis genome is characterized by an inherently complex organization, since it has undergone ancient whole genome duplications, followed by gene reduction, diploidization events and extended rearrangements, which relocated and split up the retained portions. These events, together with probable chromosome reductions, dramatically increased the genome complexity, limiting its role as a reference. The identification of paralogs and single copy genes within a highly duplicated genome is a prerequisite to understand its organization and evolution and to improve its exploitation in comparative genomics. This is still controversial, even in the widely studied Arabidopsis genome. This is also due to the lack of a reference bioinformatics pipeline that could exhaustively identify paralogs and singleton genes. We describe here a complete computational strategy to detect both duplicated and single copy genes in a genome, discussing all the methodological issues that may strongly affect the results, their quality and their reliability. This approach was used to analyze the organization of Arabidopsis nuclear protein coding genes, and besides classifying computationally defined paralogs into networks and single copy genes into different classes, it unraveled further intriguing aspects concerning the genome annotation and the gene relationships in this reference plant species. Since our results may be useful for comparative genomics and genome functional analyses, we organized a dedicated web interface to make them accessible to the scientific community.

  20. Reference Gene Validation for Quantitative PCR Under Various Biotic and Abiotic Stress Conditions in Toxoptera citricida (Hemiptera, Aphidiae).

    PubMed

    Shang, Feng; Wei, Dan-Dan; Jiang, Xuan-Zhao; Wei, Dong; Shen, Guang-Mao; Feng, Ying-Cai; Li, Ting; Wang, Jin-Jun

    2015-08-01

    The regulation of mRNA expression level is critical for gene expression studies. Currently, quantitative reverse transcription polymerase chain reaction (qRT-PCR) is commonly used to investigate mRNA expression level of genes under various experimental conditions. An important factor that determines the optimal quantification of qRT-PCR data is the choice of the reference gene for normalization. To advance gene expression studies in Toxoptera citricida (Kirkaldy), an important citrus pest and a main vector of the Citrus tristeza virus, we used five tools (GeNorm, NormFinder, BestKeeper, ΔCt methods, and RefFinder) to evaluate seven candidate reference genes (elongation factor-1 alpha [EF1α], beta tubulin [β-TUB], 18S ribosomal RNA [18S], RNA polymerase II large subunit (RNAP II), beta actin (β-ACT), alpha tubulin, and glyceraldhyde-3-phosphate dehydrogenase) under different biotic (developmental stages and wing dimorphism) and abiotic stress (thermal, starvation, and UV irradiation) conditions. The results showed that EF1α and 18S were the most stable genes under various biotic states, β-ACT and β-TUB during thermal stress, EF1α and RNAP II under starvation stress,