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Leducq-Network for Vascular MicroRNAs (MIRVAD)
Vascular system assessed by microCT
picture kindly provided by Zhen Zhuang, Yale University
The following platform technologies are available and jointly used within the members working together within the “www.vascularmicrorna.com” community. For detailed information please see the respective member lab homepages.
RNA deep sequencing
Total RNA is isolated from samples and prepared for RNA seq to determine absolute levels of mRNA transcripts. RNA-Seq uses deep-sequencing technologies where a population of RNA (total or fractionated, such as poly(A)+) is converted to a library of cDNA fragments with adaptors attached to one or both ends. Each molecule, with or without amplification, is then sequenced in a high-throughput manner to obtain short sequences from one end (single-end sequencing) or both ends (pair-end sequencing). The reads are typically 30–400 bp, using an Illumina IG sequencers (http://medicine.yale.edu/keck/ycga/sequencing/index.aspx).
Following sequencing, the resulting reads are either aligned to a reference genome or reference transcripts, or assembled de novo without the genomic sequence to produce a genome-scale transcription map that consists of both the transcriptional structure and/or level of expression for each gene in a complex mixture. For more information please see the Sessa lab homepage.
High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP)
HITS-CLIP is a genome-wide means of mapping protein–RNA binding sites. Target identification can be performed e.g. by Argonaute-2-cross-linking immuoprecipitation (Ago2-CLIP). Briefly, we expose cells or tissue to UV-light in order to cross-link Ago2 to microRNA-targeted mRNA transcripts. Subsequently, we immunoprecipitate Ago2/transcript complexes using a monoclonal antibody, followed by treatment with RNAse to digest any areas of exposed RNA not bound to Ago2 and to eliminate non-bound transcripts. The remaining fragments, which represent the “foot-printed” regions of target transcripts where the microRNA-Ago2 complex was bound, are cloned and subjected to deep sequencing. Resulting sequence tags are then mapped to the appropriate genome and quantified to identify “peaks” of enriched binding. Computational analyses (e.g. Sylamar and MEME) are then applied to characterize microRNA binding sites. To enrich for targets of a microRNA of interest, we perform Ago2-CLIP in cells over-expressing or lacking a particular microRNA. In these cases, Ago2-CLIP fragments are enriched in or depleted of, respectively, fragments that would be bound by that particular microRNA. For more information please see the Lawson lab homepage.
Robotic-assisted functional microRNA library profiling
The Thum lab has recently successfully implemented a fully-automated robotic-assisted miRNA library screening platform for high-throughput identification of functionally relevant microRNAs. With this method several hundreds of different miRNAs can be overexpressed or silenced in basically any target cell with subsequent testing of functional consequences. For more information please see the Thum lab homepage.
The state of the art facility of the Mayr lab is focused on vascular proteomics. Examples for the technical repertoire and available systems for protein identification and quantification are Two-dimensional gel electrophoresis (2-DE), Differential in-gel electrophoresis (DIGE), Nanoflow liquid chromatography, an Orbitrap mass analyzer ( LTQ Orbitrap XL, Thermo Scientific), TSQ Vantage triple stage quadrupole mass spectrometer (Thermo Scientific). The TSQ Vantage is interfaced to a splitless nano LC system (Eksigent) and a TriVersa NanoMate, a chip-based technology from Advion that combines the strengths of liquid chromatography, mass spectrometry, fraction collection, and chip-based infusion in one integrated system.For more information please see the Mayr lab homepage
The zebrafish facility that houses >500 tanks is located immediately adjacent to the Lawson lab. Additional tank space is available in the UMass Medical School Aquatics core facility, which holds approximately 3000 2.2L and 1000 3/4L tanks. A major benefit of using the zebrafish as a model is its rapid early development. There is a separate procedure room equipped with injection apparatuses in the Aquatics Core Facility. Daily care and feeding of all zebrafish on campus is performed by the Animal Facility staff at UMassMedicalSchool. Using this model, the basic vertebrate body plan is recognizable and several organ systems are beginning to form and can be observed. Just ten hours later, the embryo will have a fully functional circulatory system which allows in depth studies of the vascular system. An additional benefit is that zebrafish embryos are fertilized and develop externally allowing easy access to embryos for the purpose of time lapse analysis and other manipulations. For more information please see the Lawson lab homepage.