The overall aim of the Molecular and Cellular Analytical (MCA) core is to support the investigation of human and non-human metabolism from a cellular/molecular perspective. The core was recently restructured to focus on investigation in three areas:
1) Lipidomics and mass spectrometry
2) Bioinformatic analysis of RNAseq data
3) Mitochondrial respiration.
This is a new core being supported by the NORC based on feedback from NORC members on campus. This core grew from the LC/MS and GC/MS analytical capabilities of the Bergman laboratory. We offer analytical services, as well as expertise in interpretation of lipidomic data.
The NORC Lipidomics/mass spectrometry core can quantitatively analyze the following lipids from plasma as well as cells and tissues (muscle, heart, liver, bone):
– Triglyceride species (number of carbons, number of double bonds)
– Diacylglycerol isomers (1,2- vs 1,3-) and species (20+ and growing)
– Dihydroceramide and ceramide species
– Glucosylceramide and lactosylceramide species
– Sphingosine, sphingosine-1-phosphate, ceramide phosphate species
– Sphingomyelin species
– Acyl-carnitine species
– Phosphatidic Acid and Lysophosphatidic Acid species
– Free fatty acid species
Dr. Bergman has been performing GC/MS analysis for his research as well as collaborative projects since 2004. They analyze plasma samples for concentration and enrichment of:
– Free fatty acids
RNAseq bioinformatic pipeline
Next-Gen Sequencing. Our purpose is to provide existing members the Nutrition and Obesity Research Center and researchers at the University of Colorado Anschutz Campus with a consolidated workflow for the management, processing, and biological interpretation of unbiased RNA sequencing gene expression data.
Basics of the Service. We process high resolution RNA sequencing data (FastQ) and implement state-of-the-art computational and bioinformatics methods to supply the end user with statistically significant genes, annotated gene lists, canonical pathways, putative upstream regulators, disease states, gene interaction networks, and biological meaning specific to their research question.
Previous Experience. We have nearly two decades each of global mRNA expression analysis experience. We have a well-established and comprehensive pipeline to quantify significant and meaningful gene expression changes present in your RNA sequencing experiments.
The Mitochondrial function sub core of the MCA is a use of equipment core to facilitate the comprehensive measurement of mitochondrial function and cellular bioenergetics for both academic and industry users. In contrast to the other 2 subcores of the MCA, the mitochondria subcore provides the equipment rather than services for end users, thereby making the various methodologies more cost effective, and facilitating training and professional development in the use and understanding of the various respiration methodologies. Respirometry measurements may be performed on a host of biological samples including cultured/primary cells, permeabilized fibers or cells, and or isolated mitochondria. Both the XFe96 Seahorse and the Oroboros O2k instruments have advantages, as well as limitations to their use. In following, the inclusion of a number of differing instruments within the Mitochondrial function subcore provide researchers the capacity for a more comprehensive methodological and systematic in depth approach to cellular and mitochondrial specific respirometry studies if needed.
The following major pieces of equipment for the mitochondrial function sub core are:
- XFe96 Seahorse Extracellular Flux Analyzer,
- Biotek Cytation-1 cell imaging system,
- Oroboros O2k Respirometer,
- Oroboros O2k FluoRespirometer which allows for the simultaneous monitoring of oxygen consumption together with either ROS production or mitochondrial membrane potential,
- 2 Strathkelvin respirometers coupled to a 782 oxygen meter,
- Protein Simple Jess
The following equipment is also available for use for studies on mitochondrial function if needed: High speed refrigerated floor centrifuge, 2 bench top centrifuges, microfuges, Mettler semi-micro balances, dissecting microscope.
Both an XFe96 Seahorse and a pair of O2k Respirometers are available for respirometry assays in intact cells, permeabilized cells, and isolated mitochondria. These assays can be used to quantify mitochondrial ATP production, maximal capacity of the respiratory chain, uncoupling, mitochondrial membrane potential, and ROS production.
Basic training/consultation is routinely provided for use of the Seahorse XF96 Extracellular Flux Analyzer, and the BioTek Cytation instruments. In contrast, more in depth training/consultation is provided for the Oroboros O2k, and Strathkelvin systems.
Normalization of the XFe96 Seahorse data via cell number, genomic DNA, and total cellular protein, can be a time consuming processes. To facilitate a simple, time saving solution to normalization, the NORC MCA core has purchased a BioTek Cytation-1 Cell Imaging Multi-Mode Reader to work in concert with the XFe96 Seahorse for normalization purposes. Following a Seahorse assay run, Hoechst stain is added, and the Seahorse plate is then transferred to the Cytation-1 imager. Images and counts are then transferred the back into the Wave software for normalization of the Seahorse ECAR and OCAR data. The entire normalization procedure takes approximately 30min.
Mitochondrial proteins are essential for mitochondrial function. In following, the NORC MCA core has purchased a Protein Simple Jess. The Jess is a fully automated capillary-based immunoassay method, requiring less sample, antibody, and time compared to traditional western blots. Simply pipette samples and reagents into the microplate, and the machine automatically separates proteins by size, manages antibody additions, incubations, washes, and detection. Up to 25 samples may be run in approximately 3hrs. Multiplexing is also possible, as the Jess allows for 4 different ways to analyze proteins, Fluorescent (IR and NIR), chemiluminescent, and protein normalization.
Scheduling: In order to schedule/reserve either the Seahorse/Cytation or the Jess you will need an iLabs account. If you do not have one, you can set up an account through the following link: https://cu.corefacilities.org/account/login.
For any additional inquiries, contact: Matthew.firstname.lastname@example.org
Dr. Matt Jackman: MATTHEW.JACKMAN@CUANSCHUTZ.EDU
Dr. Kartik Shankar: KARTIK.SHANKAR@CUANSCHUTZ.EDU
Dr. Bryan Bergman: BRYAN.BERGMAN@CUANSCHUTZ.EDU