Histopathology is the main connecting cross-disciplinary subject in medicine, and enables the direct visualization of tissue changes. This technique has the unique capacity to analyze a marker at the single cell/tissue level while preserving the morphological context. Formalin-fixed and paraffin-embedded (FFPE) human as well as mouse material will be used for a morphology-based description of the different tissue components using histochemical, immunohistochemical, as well as in situ, technologies (RNAscope).
Immunohistochemistry (IHC) allows the detection of cell-associated or non-cell fixed proteins, modern in situ hybridization (ISH) technologies enable the reliable and specific detection of mRNA, miRNA, and lncRNA within the tissue environment context. Dual ISH and IHC stainings will be used for the identification of cytokines and their cellular origin. Furthermore, the Nanostring technology enables the expression profiling of more than 500 inflammation- and immunology-related genes on human or mouse FFPE material.
- Histopathology: Standard tissue preparation on FFPE material or frozen material using different histochemical stainings.
- Immunohistochemistry: Standard detection of different proteins on-slides in the preserved tissue context.
- RNAScope: Specific detection of mRNA, miRNA, and lncRNA within the tissue environment,
- Nanostring platform: Inflammation- and immunology-related expression profiling of more than 500 genes on human or mouse FFPE material.
Internal resources: Immunohistochemical detection platforms (Bond MAX by Leica, Benchmark by Ventana, Dako Stainer); RNAscope platform including dual ISH-IHC staining (Ventana Discovery XT); Nanostring platform.
Connection of this CF to specific projects: This CF will be required and utilized in projects A (Baldus), B (Benzing), E (Pasparakis), F (Rosenkranz), G (Rudolph), and I (Trifunovic).
MS-based proteomics is routinely used to investigate the composition and dynamics of cellular organelles, protein complexes, and signaling pathways. Contemporary shotgun proteomics utilizes site-specific proteases to perform protein digestion and the resulting peptide mixture is then subjected to mass spectrometery. Modern high resolution mass spectrometers, such as quadrupole Orbitraps, consist of a selection quadrupole, a high-efficiency C-trap and higher-energy collisional dissociation (HCD) octopole collision cell that allows for a rapid and sensitive fragmentation of peptides.
Another challenge for proteomics is the identification of peptides with post-translational modifications (PTMs) from cells and tissues since biological relevant modifications are often of low abundance. To identify those PTMs by MS, several modification enrichment strategies were developed.
- Quantitative proteomics (using LFQ, SILAC, and ITRAQ): The proteomics facility at the CECAD uses metabolic and chemical labeling techniques for protein quantification in cell culture and living animals. Protocols and advice for metabolic and chemical labeling are available and the technical assistant of the CF will support members of the research training group with SILAC labeling, protein extraction, and protein/peptide separation.
- Protein-protein interaction studies: Protein-protein interactions are fundamental to the understanding of biological processes. This method is based on the enrichment of bait proteins using a specific antibody (endogenous level) or in the case of a tagged protein (FLAG, HA, or GFP), a tag-specific antibody that is immobilized on a surface (e.g. beads, well plates). Bait proteins and their interactors (preys) become enriched after applying several mild washing steps. The protein mixture is then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and quantified by an intensity-based label-free quantification.
- PTM mapping - Enrichment of post-translational modifications: The major challenge for the detection of PTMs is the low stoichiometry for most of the modified peptides in complex biological samples. The enrichment of phosphopeptides will be performed by high-pH reversed-phase chromatography and phosphopeptide extraction with titanium dioxide beads. For the enrichment of ubiquitination sites (ubiquitin remnants), tryptic peptides from cells/tissue will be immunoprecipitated with an ubiquitin remnant motif antibody and then analyzed by mass spectrometry.
Internal resources: The CF consists of one LTQ Orbitrap discovery and three high resolution hybrid quadrupole Orbitrap mass spectrometers (2xExactive Plus, QExactive Plus HF-X). Each instrument is connected to a nano-HPLC system and uses the electro-spray ionization technique (ESI). Gel- and liquid-based separations on the protein and peptide levels are performed using 1D-SDS PAGE, isoelectric fractionation, as well as anion exchange and reversed phase chromatography (UHPLC).
Connection of this CF to specific projects: This CF will be required and utilized in projects B (Benzing), C (Brüning), D (Papantonis), E (Pasparakis), F (Rosenkranz), H (Schumacher), and I (Trifunovic).
Molecular imaging allows the visualization of different physiological or pathophysiological processes at the molecular level with high resolution and extraordinary sensitivity. This detection requires the preparation of molecular probes that enable the non-invasive detection of small molecular alterations in preclinical models. The molecular imaging probes are injected into the animal model and interact specifically with its molecular target and specifically display the expression of, e.g. enzymes or receptor systems. A molecular imaging probe typically comprises a signal agent, a targeting moiety, and a linker connecting the targeting moiety and the signal agent. The signal agent usually produces signal for that subsequently allow imaging. A PET imaging probe requires a positron-emitting radionuclide as the label. The signal agent is a radionuclide produced at a cyclotron. These PET nuclides are then incorporated via labeling chemistry into biomolecules suitable for PET imaging. After tracer development, the novel imaging probes can be evaluated in preclinical settings. After successful biological evaluation, the probe is produced on a preparative scale using automation processes.
For the whole process, i.e. target identification, probe design, radiolabeling, preclinical evaluation, and automation, the following infrastructure is required and provided:
- Cyclotron: Radionuclide production in cooperation with Forschungszentrum Jülich.
- Radiochemistry laboratories: Radiochemical developments to prepare imaging agents.
- Small animal imaging facility: Biological in vivo and in vitro evaluation of imaging probes.
- Hot-cells and automated synthesis modules: For the production on a preparative scale, leaded-shielded hot cells are required that enable us to handle high radioactivity amounts.
Connection of this CF to specific projects: This CF will be required and utilized in projects A (Baldus), E (Pasparakis), F (Rosenkranz), and G (Rudolph).
Next generation sequencing (NGS) methods are currently revolutionizing the field of genome research. They enable researchers to decode DNA and RNA sequences at an unprecedented depth and level of detail. The Cologne Center for Genomics set up cutting-edge NGS technologies to assist all life scientists of the University of Cologne in their bio-medical research efforts.
- Genomics – Whole genome shotgun sequencing (WGS): Random fragments of a complete genome are sequenced using PCR-free library preparation. Standard pipelines for alignment against a reference sequence and variant detection are available. De novo sequencing: random fragments of up to 10kb original size are sequenced. Standard pipelines include de novo assembly and annotation. Non-sequencing-based genome mapping and linked-read technologies are used to improve the results. Structural variations, e. g. in cancer cells, can be analyzed with similar methods and can be validated using DNA combing technology. Targeted resequencing, including whole-exome sequencing (WES): Random fragments of a selected genome region are sequenced using various enrichment technologies. Standard pipelines for alignment against a reference and variant detection are available. ATAC-seq and HiC methods are used to detect changes in chromatin conformation.
- Transcriptomics – RNA-seq: Reverse transcribed cDNA fragments from total RNA of any organism are sequenced. Standard analyses include alignment against a transcript database, detection of splice sites and gene expression estimation. Different approaches exist for different needs like mRNA-seq using Poly-A selection, total RNA-seq using hybridization based ribo-depletion protocols or 3´mRNA-seq for simple and cost effective expression profiling. Low-input RNA protocols are available for 10pg-10ng and 500pg-100ng starting material. Single cell RNA sequencing (scRNA-seq) is supported with two different platforms based on either nano-dispensing or micro-droplet technologies.
- Epigenomics – ChIP-seq/RIP-seq: DNA or RNA fragments extracted by chromatin immunoprecipitation are sequenced. Standard analysis pipelines include alignment against a reference genome, detection of binding sites (peaks) and normalization against a control. Small RNA-seq: Reverse transcribed cDNA fragments of size-selected RNA molecules are sequenced. Standard analyses include expression comparison, sequence variation assessment and prediction of novel micro RNAs. WGBS: Bisulphid treated DNA is sequenced to allow for detection of DNA; ethylation in any organism.
Internal resources: The core of the facility consists of Illumina’s HiSeq4000, HiSeq2000, Miseq sequencers. Further Illumina iScan and Affymetrix Gene Titan array platforms are in place. Diverse robotic systems from Beckman, Hamilton, Tecan and Agilent are in use for automated protocols. ABI 3730 Capillary Sequencers Applied Biosystems are used for validation/segregation studies. Further equipment comprises numerous PCR cyclers and qPCR divices from Applied Biosystems, Diagenode’s Bioruptor, 2200 Tape Stations from Agilent, PeqLap’s NanoDrop, and Invitrogen’s Qubit. For cfDNA studies the Qiagen PSQ96HS (Pyrosequencer) and Applied Biosystems’ QuantStudio® 3D Digital PCR System are available. Dedicated equipment for genomic mapping /SV detection includes BioNano’s Irys System and the Fiber Vision System from Genomic Vision. For scRNA-seq the ICELL8 System from Wafergen and the Chromium™ Controller from 10x Genomics were recently acquired.
Connection of this CF to specific projects: This CF will be required and utilized in projects B (Benzing), D (Papantonis), H (Schumacher), E (Pasparakis), and I (Trifunovic).