Protocol: A simple phenol-based method for 96-well extraction of high quality RNA from Arabidopsis

Background: Many experiments in modern plant molecular biology require the processing of large numbers of samples for a variety of applications from mutant screens to the analysis of natural variants. A severe bottleneck to many such analyses is the acquisition of good yields of high quality RNA suitable for use in sensitive downstream applications such as real time quantitative reverse-transcription-polymerase chain reaction (real time qRT-PCR). Although several commercial kits are available for high-throughput RNA extraction in 96-well format, only one non-kit method has been described in the literature using the commercial reagent TRIZOL.Results: We describe an unusual phenomenon when using TRIZOL reagent with young Arabidopsis seedlings. This prompted us to develop a high-throughput RNA extraction protocol (HTP96) adapted from a well established phenol:chloroform-LiCl method (P:C-L) that is cheap, reliable and requires no specialist equipment. With this protocol 192 high quality RNA samples can be prepared in 96-well format in three hours (less than 1 minute per sample) with less than 1% loss of samples. We demonstrate that the RNA derived from this protocol is of high quality and suitable for use in real time qRT-PCR assays.Conclusion: The development of the HTP96 protocol has vastly increased our sample throughput, allowing us to fully exploit the large sample capacity of modern real time qRT-PCR thermocyclers, now commonplace in many labs, and develop an effective high-throughput gene expression platform. We propose that the HTP96 protocol will significantly benefit any plant scientist with the task of obtaining hundreds of high quality RNA extractions

The C. elegans HP1 homologue HPL-2 and the LIN-13 zinc finger protein form a complex implicated in vulval development.

International audienceHP1 proteins are essential components of heterochromatin and contribute to the transcriptional repression of euchromatic genes via the recruitment to specific promoters by corepressor proteins including TIF1 and Rb. The Caenorhabditis elegans HP1 homologue HPL-2 acts in the "synMuv" (synthetic multivulval) pathway, which defines redundant negative regulators of a Ras signaling cascade required for vulval induction. Several synMuv genes encode for chromatin-associated proteins involved in transcriptional regulation, including Rb and components of the Mi-2/NuRD and TIP60/NuA4 chromatin remodeling complexes. Here, we show that HPL-2 physically interacts in vitro and in vivo with the multiple zinc finger protein LIN-13, another member of the synMuv pathway. A variant of the conserved PXVXL motif found in many HP1-interacting proteins mediates LIN-13 binding to the CSD of HPL-2. We further show by in vivo localization studies that LIN-13 is required for HPL-2 recruitment in nuclear foci. Our data suggest that the LIN-13/HPL-2 complex may physically link a subset of the Rb related synMuv proteins to chromatin

Face au changement climatique, quelles stratégies d’atténuation et d’adaptation pour les productions avicoles ?

La viande de volaille et les œufs sont des sources principales de protéines animales pour l'alimentation humaine dans le monde. Leur production a augmenté rapidement au cours des dernières décennies. Cependant, les productions avicoles sont vulnérables au changement climatique, en particulier au réchauffement de la planète et à ses conséquences directes et indirectes. Pour y faire face, il est nécessaire de mettre en place des stratégies d'adaptation des animaux, en particulier en améliorant leur résilience. Ces stratégies nécessitent d’une part de mieux comprendre la physiologie des oiseaux (thermorégulation, efficacité pour la production de viande et d'œufs…) et d’autre part de rechercher des innovations en lien avec la nutrition, la santé, la reprogrammation précoce ou encore la génétique (intégration de nouveaux caractères adaptatifs dans les stratégies de sélection). Il faut également trouver des solutions au niveau des systèmes de production, par exemple en prenant en compte les aires de répartition géographique des maladies liées au changement climatique et en introduisant des pratiques d'atténuation pour réduire les consommations d'énergie et les émissions de gaz à effet de serre. Des recherches interdisciplinaires axées sur la génétique, les méthodes techniques (telles que la programmation thermique précoce), les solutions d'ingénierie, des innovations nutritionnelles et de nouvelles stratégies d'élevage agroécologiques sont ainsi développées. Ces stratégies tiennent compte de la demande sociale croissante en faveur de productions animales éthiques dans les perspectives d’une seule santé (« One Health ») et d’un seul bien-être (« One Welfare ») et visent à limiter la concurrence entre l'homme et les animaux pour les ressources alimentaires. Cette revue illustre par quelques exemples les leviers d'amélioration et de stratégies adaptatives envisageables pour rendre les animaux et les systèmes de production avicole plus résilients dans le contexte du changement climatique

First landscape of binding to chromosomes for a domesticated mariner transposase in the human genome: diversity of genomic targets of SETMAR isoforms in two colorectal cell lines

Setmar is a 3-exons gene coding a SET domain fused to a Hsmar1 transposase. Its different transcripts theoretically encode 8 isoforms with SET moieties differently spliced. In vitro, the largest isoform binds specifically to Hsmar1 DNA ends and with no specificity to DNA when it is associated with hPso4. In colon cell lines, we found they bind specifically to two chromosomal targets depending probably on the isoform, Hsmar1 ends and sites with no conserved motifs. We also discovered that the isoforms profile was different between cell lines and patient tissues, suggesting the isoforms encoded by this gene in healthy cells and their functions are currently not investigated

Tissue Resources for the Functional Annotation of Animal Genomes

In order to generate an atlas of the functional elements driving genome expression in domestic animals, the Functional Annotation of Animal Genome (FAANG) strategy was to sample many tissues from a few animals of different species, sexes, ages, and production stages. This article presents the collection of tissue samples for four species produced by two pilot projects, at INRAE (National Research Institute for Agriculture, Food and Environment) and the University of California, Davis. There were three mammals (cattle, goat, and pig) and one bird (chicken). It describes the metadata characterizing these reference sets (1) for animals with origin and selection history, physiological status, and environmental conditions; (2) for samples with collection site and tissue/cell processing; (3) for quality control; and (4) for storage and further distribution. Three sets are identified: set 1 comprises tissues for which collection can be standardized and for which representative aliquots can be easily distributed (liver, spleen, lung, heart, fat depot, skin, muscle, and peripheral blood mononuclear cells); set 2 comprises tissues requiring special protocols because of their cellular heterogeneity (brain, digestive tract, secretory organs, gonads and gametes, reproductive tract, immune tissues, cartilage); set 3 comprises specific cell preparations (immune cells, tracheal epithelial cells). Dedicated sampling protocols were established and uploaded in https://data.faang.org/protocol/samples. Specificities between mammals and chicken are described when relevant. A total of 73 different tissues or tissue sections were collected, and 21 are common to the four species. Having a common set of tissues will facilitate the transfer of knowledge within and between species and will contribute to decrease animal experimentation. Combining data on the same samples will facilitate data integration. Quality control was performed on some tissues with RNA extraction and RNA quality control. More than 5,000 samples have been stored with unique identifiers, and more than 4,000 were uploaded onto the Biosamples database, provided that standard ontologies were available to describe the sample. Many tissues have already been used to implement FAANG assays, with published results. All samples are available without restriction for further assays. The requesting procedure is described. Members of FAANG are encouraged to apply a range of molecular assays to characterize the functional status of collected samples and share their results, in line with the FAIR (Findable, Accessible, Interoperable, and Reusable) data principles

STRESSEPIMARK : Suivi de la réaction immédiate au stress thermique et de sa mémorisation dans les biotechnologies aviaires embryonnaires

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Que peut-on faire avec l’épigénétique ?

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Utilisation de la thermographie infrarouge chez la caille japonaise

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QuailHeatE: Effets transgénérationnels d'une exposition précoce à la chaleur chez la caillejaponaise

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