Gene expression studies in different genotypes of an ectomycorrhizal fungus require a high number of reliable reference genes

Quantitative reverse transcription PCR (qRT-PCR) has become the standard technique for the expression analysis of a set of chosen genes of interest. The accuracy and reliability of qRT-PCR measurements strongly depends on the normalization with appropriate endogenous reference genes. In this study a set of candidate reference genes for the use in gene expression studies of a basidiomycete fungus, Suillus luteus, exposed to toxic concentrations of zinc or cadmium was identified, evaluated and validated. Seven candidate genes were selected from cDNA-AFLP as stably expressed and the algorithms geNorm and Normfinder were used to evaluate these genes alongside the traditionally used housekeeping genes (actin, tubulin) in different S. luteus isolates. The use of several S. luteus isolates revealed that each isolate has its own most stably expressed set of reference genes, regardless of the metal treatments, in casu metal exposures. Metal treatments had only a minor impact on the expression of the candidate reference genes. The validated reference genes outperform the in fungal research commonly used single, arbitrary chosen (“housekeeping”) genes in terms of reliability, and have the potential to be suitable reference genes when studying the effect of other environmental factors. A relatively high number of reference genes is required to correct for intraspecific variability when studying natural populations.qRT-PCR normalization; gene expression; Suillus; heavy metal tolerance; intraspecific variatio

Gene expression studies in different genotypes of an ectomycorrhizal fungus require a 1 high number of reliable reference genes. 2 3

Abstract 13 Quantitative reverse transcription PCR (qRT-PCR) has become the standard technique for the 14 expression analysis of a set of chosen genes of interest. The accuracy and reliability of qRT-15 PCR measurements strongly depends on the normalization with appropriate endogenous 16 reference genes. In this study a set of candidate reference genes for the use in gene expression 17 studies of a basidiomycete fungus, Suillus luteus, exposed to toxic concentrations of zinc or 18 cadmium was identified, evaluated and validated. Seven candidate genes were selected from 19 cDNA-AFLP as stably expressed and the algorithms geNorm and Normfinder were used to 20 evaluate these genes alongside the traditionally used housekeeping genes (actin, tubulin) in 21 different S. luteus isolates. The use of several S. luteus isolates revealed that each isolate has 22 its own most stably expressed set of reference genes, regardless of the metal treatments, i

Inoculation with Rhizobium etli enhances organic acid exudation in common bean (Phaseolus vulgaris L.) subjected to phosphorus deficiency

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A systematic review of correlates of physical activity in patients with schizophrenia

OBJECTIVE: The present review evaluates systematically the published quantitative studies of correlates of PA in patients with schizophrenia. METHOD: EMBASE, PsycINFO, PubMed, ISI Web of Science, CINAHL and PEDro were searched from their inception to 1 July 2011 combining the medical subject heading 'schizophrenia' with 'physical activity' or 'physical inactivity' or 'exercise' or 'health education' or 'health behaviour' or 'health promotion'. RESULTS: Out of 68 potentially eligible studies, 25 papers (n = 25,013) evaluating 36 correlates were included. Correlates consistently associated with lower PA participation are the presence of negative symptoms and cardio-metabolic comorbidity. Also, side-effects of antipsychotic medication, lack of knowledge on cardiovascular disease risk factors, no belief in the health benefits, a lower self-efficacy, other unhealthy lifestyle habits and social isolation correlated with lower PA participation. The quality of the PA measurement was not related to the proportion of significant associations (χ(2) = 3.8, P = 0.07). Current gaps in literature that need to be examined more in detail are the role of environmental and policy-level factors on PA participation in patients with schizophrenia. CONCLUSION: All correlates should be confirmed in prospective studies, and interventions to improve the modifiable variables should be developed and evaluated.status: publishe

Reliable Gene Expression Analysis by Reverse Transcription-Quantitative PCR: Reporting and Minimizing the Uncertainty in Data Accuracy

Reverse transcription-quantitative PCR (RT-qPCR) has been widely adopted to measure differences in mRNA levels; however, biological and technical variation strongly affects the accuracy of the reported differences. RT-qPCR specialists have warned that, unless researchers minimize this variability, they may report inaccurate differences and draw incorrect biological conclusions. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines describe procedures for conducting and reporting RT-qPCR experiments. The MIQE guidelines enable others to judge the reliability of reported results; however, a recent literature survey found low adherence to these guidelines. Additionally, even experiments that use appropriate procedures remain subject to individual variation that statistical methods cannot correct. For example, since ideal reference genes do not exist, the widely used method of normalizing RT-qPCR data to reference genes generates background noise that affects the accuracy of measured changes in mRNA levels. However, current RT-qPCR data reporting styles ignore this source of variation. In this commentary, we direct researchers to appropriate procedures, outline a method to present the remaining uncertainty in data accuracy, and propose an intuitive way to select reference genes to minimize uncertainty. Reporting the uncertainty in data accuracy also serves for quality assessment, enabling researchers and peer reviewers to confidently evaluate the reliability of gene expression data

Soil–plant relationships of metals and metalloids

Nutrient uptake by plants is essential for their development and for the passage of minerals into the food chain, but it also faces several limitations. Whereas soil physicochemical characteristics impose limiting factors on element availability for plants, excess of non-essential metals and metalloids pose a threat for plant health and the environment. To improve nutrient uptake, the plant possesses several mechanisms to explore the soil for minerals such as root development, but the symbiosis with microorganisms clearly improves the ability of plants to overcome these limitations. After metal uptake by the plants, plants make use of different strategies to maintain the metal homeostasis and to limit the metal-induced cellular damage. Also in the research on metal phytotoxicity, microorganisms are shown to be important players in the protection of the plant to excess metal exposure

ALTERNATIVE OXIDASE1a modulates the oxidative challenge during moderate Cd exposure in Arabidopsis thaliana leaves

This study aims to unravel the functional significance of alternative oxidase1a (AOX1a) induction in Arabidopsis thaliana leaves exposed to cadmium (Cd) by comparing wild-type (WT) plants and aox1a knockout mutants. In the absence of AOX1a, differences in stress-responsive transcript and glutathione levels suggest an increased oxidative challenge during moderate (5 mu M) and prolonged (72 h) Cd exposure. Nevertheless, aox1a knockout leaves showed lower hydrogen peroxide (H2O2) accumulation as compared to the WT due to both acute (24 h) and prolonged (72 h) exposure to 5 mu M Cd, but not to 10 mu M Cd. Taken together, we propose a working model where AOX1a acts early in the response to Cd and activates or maintains a mitochondrial signalling pathway impacting on cellular antioxidative defence at the post-transcriptional level. This fine-tuning pathway is suggested to function during moderate (5 mu M) Cd exposure while being overwhelmed during more severe (10 mu M) Cd stress. Within this framework, ethylene is required - either directly or indirectly via NADPH oxidase isoform C - to fully induce AOX1 expression. In addition, reciprocal crosstalk between these components was demonstrated in leaves of A. thaliana plants exposed to Cd