Normalization for triple-target microarray experiments

<p>Abstract</p> <p>Background</p> <p>Most microarray studies are made using labelling with one or two dyes which allows the hybridization of one or two samples on the same slide. In such experiments, the most frequently used dyes are <it>Cy</it>3 and <it>Cy</it>5. Recent improvements in the technology (dye-labelling, scanner and, image analysis) allow hybridization up to four samples simultaneously. The two additional dyes are <it>Alexa</it>488 and <it>Alexa</it>494. The triple-target or four-target technology is very promising, since it allows more flexibility in the design of experiments, an increase in the statistical power when comparing gene expressions induced by different conditions and a scaled down number of slides. However, there have been few methods proposed for statistical analysis of such data. Moreover the lowess correction of the global dye effect is available for only two-color experiments, and even if its application can be derived, it does not allow simultaneous correction of the raw data.</p> <p>Results</p> <p>We propose a two-step normalization procedure for triple-target experiments. First the dye bleeding is evaluated and corrected if necessary. Then the signal in each channel is normalized using a generalized lowess procedure to correct a global dye bias. The normalization procedure is validated using triple-self experiments and by comparing the results of triple-target and two-color experiments. Although the focus is on triple-target microarrays, the proposed method can be used to normalize <it>p </it>differently labelled targets co-hybridized on a same array, for any value of <it>p </it>greater than 2.</p> <p>Conclusion</p> <p>The proposed normalization procedure is effective: the technical biases are reduced, the number of false positives is under control in the analysis of differentially expressed genes, and the triple-target experiments are more powerful than the corresponding two-color experiments. There is room for improving the microarray experiments by simultaneously hybridizing more than two samples.</p

Multi-isotope analysis of bone collagen of Late Pleistocene ungulates reveals niche partitioning and behavioural plasticity of reindeer during MIS 3

Acknowledgements This research was funded by a Leverhulme Research Project Grant (ref: RPG-2017-410 to K.B.), and the Max Planck Institute for Evolutionary Anthropology. A Philip Leverhulme Prize (ref: PLP-2019-284 to K.B.) provided support to K.B. during the production of this manuscript, and M.S. is funded by the NWO Dutch Research council (VICI award VI.C.191.07). We thank C.-H. Bachelier and Jaques Bachelier for facilitating and supporting research at Les Cottés, and the French Ministry of Culture for allowing and funding research at Les Cottés. Thanks to Sven Steinbrenner and Annabell Reiner (MPI-EVA), and Orsolya Czére (Aberdeen) for laboratory assistance, and to Jovita Fawcett and Eléa Gutierrez (Aberdeen) for proof reading and assistance with images.Peer reviewedPublisher PD

Genome-wide interacting effects of sucrose and herbicide-mediated stress in Arabidopsis thaliana: novel insights into atrazine toxicity and sucrose-induced tolerance

<p>Abstract</p> <p>Background</p> <p>Soluble sugars, which play a central role in plant structure and metabolism, are also involved in the responses to a number of stresses, and act as metabolite signalling molecules that activate specific or hormone-crosstalk transduction pathways. The different roles of exogenous sucrose in the tolerance of <it>Arabidopsis thaliana </it>plantlets to the herbicide atrazine and oxidative stress were studied by a transcriptomic approach using CATMA arrays.</p> <p>Results</p> <p>Parallel situations of xenobiotic stress and sucrose-induced tolerance in the presence of atrazine, of sucrose, and of sucrose plus atrazine were compared. These approaches revealed that atrazine affected gene expression and therefore seedling physiology at a much larger scale than previously described, with potential impairment of protein translation and of reactive-oxygen-species (ROS) defence mechanisms. Correlatively, sucrose-induced protection against atrazine injury was associated with important modifications of gene expression related to ROS defence mechanisms and repair mechanisms. These protection-related changes of gene expression did not result only from the effects of sucrose itself, but from combined effects of sucrose and atrazine, thus strongly suggesting important interactions of sucrose and xenobiotic signalling or of sucrose and ROS signalling.</p> <p>Conclusion</p> <p>These interactions resulted in characteristic differential expression of gene families such as ascorbate peroxidases, glutathione-S-transferases and cytochrome P450s, and in the early induction of an original set of transcription factors. These genes used as molecular markers will eventually be of great importance in the context of xenobiotic tolerance and phytoremediation.</p

Lamin A/C–mediated neuromuscular junction defects in Emery-Dreifuss muscular dystrophy

The LMNA gene encodes lamins A and C, two intermediate filament-type proteins that are important determinants of interphase nuclear architecture. Mutations in LMNA lead to a wide spectrum of human diseases including autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD), which affects skeletal and cardiac muscle. The cellular mechanisms by which mutations in LMNA cause disease have been elusive. Here, we demonstrate that defects in neuromuscular junctions (NMJs) are part of the disease mechanism in AD-EDMD. Two AD-EDMD mouse models show innervation defects including misexpression of electrical activity–dependent genes and altered epigenetic chromatin modifications. Synaptic nuclei are not properly recruited to the NMJ because of mislocalization of nuclear envelope components. AD-EDMD patients with LMNA mutations show the same cellular defects as the AD-EDMD mouse models. These results suggest that lamin A/C–mediated NMJ defects contribute to the AD-EDMD disease phenotype and provide insights into the cellular and molecular mechanisms for the muscle-specific phenotype of AD-EDMD

Analysis of CATMA transcriptome data identifies hundreds of novel functional genes and improves gene models in the Arabidopsis genome

<p>Abstract</p> <p>Background</p> <p>Since the finishing of the sequencing of the <it>Arabidopsis thaliana </it>genome, the Arabidopsis community and the annotator centers have been working on the improvement of gene annotation at the structural and functional levels. In this context, we have used the large CATMA resource on the Arabidopsis transcriptome to search for genes missed by different annotation processes. Probes on the CATMA microarrays are specific gene sequence tags (GSTs) based on the CDS models predicted by the Eugene software. Among the 24 576 CATMA v2 GSTs, 677 are in regions considered as intergenic by the TAIR annotation. We analyzed the cognate transcriptome data in the CATMA resource and carried out data-mining to characterize novel genes and improve gene models.</p> <p>Results</p> <p>The statistical analysis of the results of more than 500 hybridized samples distributed among 12 organs provides an experimental validation for 465 novel genes. The hybridization evidence was confirmed by RT-PCR approaches for 88% of the 465 novel genes. Comparisons with the current annotation show that these novel genes often encode small proteins, with an average size of 137 aa. Our approach has also led to the improvement of pre-existing gene models through both the extension of 16 CDS and the identification of 13 gene models erroneously constituted of two merged CDS.</p> <p>Conclusion</p> <p>This work is a noticeable step forward in the improvement of the Arabidopsis genome annotation. We increased the number of Arabidopsis validated genes by 465 novel transcribed genes to which we associated several functional annotations such as expression profiles, sequence conservation in plants, cognate transcripts and protein motifs.</p

Expression variation in connected recombinant populations of Arabidopsis thaliana highlights distinct transcriptome architectures

<p>Abstract</p> <p>Background</p> <p>Expression traits can vary quantitatively between individuals and have a complex inheritance. Identification of the genetics underlying transcript variation can help in the understanding of phenotypic variation due to genetic factors regulating transcript abundance and shed light into divergence patterns. So far, only a limited number of studies have addressed this subject in Arabidopsis, with contrasting results due to dissimilar statistical power. Here, we present the transcriptome architecture in leaf tissue of two RIL sets obtained from a connected-cross design involving 3 commonly used accessions. We also present the transcriptome architecture observed in developing seeds of a third independent cross.</p> <p>Results</p> <p>The utilisation of the novel R/eqtl package (which goal is to automatize and extend functions from the R/qtl package) allowed us to map 4,290 and 6,534 eQTLs in the Cvi-0 × Col-0 and Bur-0 × Col-0 recombinant populations respectively. In agreement with previous studies, we observed a larger phenotypic variance explained by eQTLs in linkage with the controlled gene (potentially <it>cis</it>-acting), compared to distant loci (acting necessarily indirectly or in <it>trans</it>). Distant eQTLs hotspots were essentially not conserved between crosses, but instead, cross-specific. Accounting for confounding factors using a probabilistic approach (VBQTL) increased the mapping resolution and the number of significant associations. Moreover, using local eQTLs obtained from this approach, we detected evidence for a directional allelic effect in genes with related function, where significantly more eQTLs than expected by chance were up-regulated from one of the accessions. Primary experimental data, analysis parameters, eQTL results and visualisation of LOD score curves presented here are stored and accessible through the QTLstore service database <url>http://qtlstore.versailles.inra.fr/</url>.</p> <p>Conclusions</p> <p>Our results demonstrate the extensive diversity and moderately conserved eQTL landscape between crosses and validate the utilisation of expression traits to explore for candidates behind phenotypic variation among accessions. Furthermore, this stresses the need for a wider spectrum of diversity to fully understand expression trait variation within a species.</p

Sélection de variables pour la classification par mélanges gaussiens pour prédire la fonction des gènes orphelins

Biologists are interested in predicting the gene functions of sequenced genome organisms according to microarray transcriptome data. The microarray technology development allows one to study the whole genome in different experimental conditions. The information abundance may seem to be an advantage for the gene clustering. However, the structure of interest can often be contained in a subset of the available variables. The currently available variable selection procedures in model-based clustering assume that the irrelevant clustering variables are all independent or are all linked with the relevant clustering variables. A more versatile variable selection model is proposed, taking into account three possible roles for each variable: The relevant clustering variables, the redundant variables and the independent variables. A model selection criterion and a variable selection algorithm are derived for this new variable role modelling. The interest of this new modelling for discovering the function of orphan genes is highlighted on a transcriptome dataset for the arabidopsis thaliana plant.Les biologistes s’attachent actuellement à prédire la fonction des gènes d’organismes de génome séquence à partir de données transcriptomes, issues de l’utilisation des puces à ADN. Le d´développement de cette technologie permet de tester l’expression de l’ensemble du génome dans de nombreuses conditions expérimentales. Cette quantité d’information peut alors sembler être un atout pour la classification des gènes. Pourtant il est courant que seul un sous-ensemble contienne l’information pertinente pour la classification. Les procédures de sélection des variables en classification non supervisée par mélanges gaussiens supposent généralement que les variables non informatives pour la classification sont soit toutes indépendantes, soit liées à des variables informatives. Nous proposons une nouvelle modélisation du rôle des variables plus polyvalente : les variables sont soit informatives pour la classification, soit redondantes, soit totalement indépendantes. Nous proposons un critère de sélection des variables et un algorithme pour cette nouvelle modélisation. L’intérêt de cette nouvelle modélisation pour la prédiction de la fonction des gènes orphelins est illustrée sur un ensemble de données transcriptomes obtenues chez Arabidopsis thaliana

Brain transcriptional stability upon prion protein-encoding gene invalidation in zygotic or adult mouse

<p>Abstract</p> <p>Background</p> <p>The physiological function of the prion protein remains largely elusive while its key role in prion infection has been expansively documented. To potentially assess this conundrum, we performed a comparative transcriptomic analysis of the brain of wild-type mice with that of transgenic mice invalidated at this locus either at the zygotic or at the adult stages.</p> <p>Results</p> <p>Only subtle transcriptomic differences resulting from the <it>Prnp </it>knockout could be evidenced, beside <it>Prnp </it>itself, in the analyzed adult brains following microarray analysis of 24 109 mouse genes and QPCR assessment of some of the putatively marginally modulated loci. When performed at the adult stage, neuronal <it>Prnp </it>disruption appeared to sequentially induce a response to an oxidative stress and a remodeling of the nervous system. However, these events involved only a limited number of genes, expression levels of which were only slightly modified and not always confirmed by RT-qPCR. If not, the qPCR obtained data suggested even less pronounced differences.</p> <p>Conclusions</p> <p>These results suggest that the physiological function of PrP is redundant at the adult stage or important for only a small subset of the brain cell population under classical breeding conditions. Following its early reported embryonic developmental regulation, this lack of response could also imply that PrP has a more detrimental role during mouse embryogenesis and that potential transient compensatory mechanisms have to be searched for at the time this locus becomes transcriptionally activated.</p

Widespread anti-sense transcription in apple is correlated with siRNA production and indicates a large potential for transcriptional and/or post-transcriptional control

Characterizing the transcriptome of eukaryotic organisms is essential for studying gene regulation and its impact on phenotype. The realization that anti-sense (AS) and noncoding RNA transcription is pervasive in many genomes has emphasized our limited understanding of gene transcription and post-transcriptional regulation. Numerous mechanisms including convergent transcription, anti-correlated expression of sense and AS transcripts, and RNAi remain ill-defined.Here, we have combined microarray analysis and high-throughput sequencing of small RNAs (sRNAs) to unravel the complexity of transcriptional and potential post-transcriptional regulation in eight organs of apple (Malus × domestica). The percentage of AS transcript expression is higher than that identified in annual plants such as rice and Arabidopsis thaliana. Furthermore, we show that a majority of AS transcripts are transcribed beyond 3′UTR regions, and may cover a significant portion of the predicted sense transcripts. Finally we demonstrate at a genome-wide scale that anti-sense transcript expression is correlated with the presence of both short (21–23 nt) and long (&gt; 30 nt) siRNAs, and that the sRNA coverage depth varies with the level of AS transcript expression. Our study provides a new insight on the functional role of anti-sense transcripts at the genome-wide level, and a new basis for the understanding of sRNA biogenesis in plants

Upregulation of PPARβ/δ Is Associated with Structural and Functional Changes in the Type I Diabetes Rat Diaphragm

Diabetes mellitus is associated with alterations in peripheral striated muscles and cardiomyopathy. We examined diaphragmatic function and fiber composition and identified the role of peroxisome proliferator-activated receptors (PPAR alpha and beta/delta) as a factor involved in diaphragm muscle plasticity in response to type I diabetes.Streptozotocin-treated rats were studied after 8 weeks and compared with their controls. Diaphragmatic strips were stimulated in vitro and mechanical and energetic variables were measured, cross bridge kinetics assessed, and the effects of fatigue and hypoxia evaluated. Morphometry, myosin heavy chain isoforms, PPAR alpha and beta/delta gene and protein expression were also assessed. Diabetes induced a decrease in maximum velocity of shortening (-14%, P<0.05) associated with a decrease in myosin ATPase activity (-49%, P<0.05), and an increase in force (+20%, P<0.05) associated with an increase in the number of cross bridges (+14%, P<0.05). These modifications were in agreement with a shift towards slow myosin heavy chain fibers and were associated with an upregulation of PPARbeta/delta (+314% increase in gene and +190% increase in protein expression, P<0.05). In addition, greater resistances to fatigue and hypoxia were observed in diabetic rats.Type I diabetes induced complex mechanical and energetic changes in the rat diaphragm and was associated with an up-regulation of PPARbeta/delta that could improve resistance to fatigue and hypoxia and favour the shift towards slow myosin heavy chain isoforms