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Metabolic Core Services and Pricing


 

Gene Expression Analysis Services

Cellular Bioenergetics Analysis Services

Protein, Hormone and Metabolite Analysis Services

Pricing

Gene Expression Analysis Services

RNA isolation

Commercially available kits are used to perform RNA isolation from various tissues and cell cultures. Depending on the source tissue, various modifications are performed with these kits to ensure the best RNA purification possible.

RNA AnalysisExperion System for RNA Analysis

The Experion automated electrophoresis system combines Bio-Rad's expertise in electrophoresis with the innovation of Caliper Life Sciences' LabChip technology to deliver high-performance, affordable automation. The system produces rapid, reproducible and reliable quantitation and qualitation of protein and nucleic acid samples.

Routine and reproducible separation of RNA samples can be accomplished in as little as 30 minutes for 12 samples with only 1.5 μl RNA sample needed.

Real Time Quantitative PCR

The iQ5 real-time PCR detection system is a thermal cycler with 96-well reaction module. With the iQ5 system, you can accurately quantitate targets over a dynamic range of more than nine orders of iQ5 Real-time PCR Detection Systemmagnitude. You can also perform qualitative assays for allelic discrimination or screening for known mutations. The iQ5 system offers five-target analysis capabilities for multiplex PCR and data collection and analysis options for gene expression analysis. The iQ5 system is compatible with multiple detection chemistries, providing maximum flexibility in real-time PCR applications.

Taqman probes and SYBR primer sets (listed hereavailable from the Core range from genes involved in gluconeogenesis and lipogenesis to neuropeptides, inflammatory markers, and transcription factors, as well as reference genes for normalization of gene expression. Custom primer sets may be designed for genes of interest not currently available in the Core.


Cellular Bioenergetics Analysis Services

The XF Extracellular Flux Analyzer is a fully-integrated instrument that simultaneously measures the two major energy yielding pathways - aerobic respiration and glycolysis - in a convenient, microplate format.XF Analyzer

This fast and sensitive measurement of cellular bioenergetics is label-free, enabling time-resolved analysis and reuse of the cells. XF instrument offers scientists the ability to: (1) simultaneously measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in as little as five minutes, (2) perform time-resolved measurements of a single population of cells over a period of minutes or hours, and (3) re-use cell samples after completing an XF measurement.

The researcher will be trained by the Core to run their own experiments and will be responsible for purchasing their own XF24 FluxPaks.
 

Protein, Hormone and Metabolite Analysis Services

Western Blotting

The Core provides services for tissue procurement/preparation and processing for analysis of phosphorylation and expression of key post-receptor insulin signaling proteins that regulate insulin sensitivity and lipid metabolism at the cellular level. Western blot analysis of the insulin receptor signaling cascade is a standard focus in the Core, including (but not limited to) IRS-1, PI 3-kinase, Akt, GSK3, JNK, TNFalpha and GLUT4 protein levels in muscle, adipose and liver tissues from human, rodent and sheep. The Core measures activity and phosphorylation state (serine/threonine) where appropriate. Analysis of insulin/IGF-1 cell signaling via mTOR, p70S6 kinase, E1F2alpha and 4E-BP1 as cellular markers for protein synthesis, including site-specific activation in tissues and cells is also available. The Core has a vast library of antibodies (listed herethat have been tested in a number of tissues and species.

ELISA and Multiplex Panels

Immunoassay of hormones including insulin, leptin, adiponectin and resistin is provided by the Core for serum/plasma/culture medium from human, mouse and rat. Multiplex panels look at a variety of cytokines, adipokines, and other markers of metabolic disease in human, mouse and rat serum/plasma/medium from cultured cells.

ELISA/EIA: Assays involve capture of the hormone of interest by antibody to that hormone bound on the surface of the wells of a microtiter plate. Each assay is specific for measuring only one hormone at a time. Sample volume of 25-100 µl is required for each assay.

MULTIPLEX PANELS: The development of a bead-based suspension assay with the ability to measure several targets of interest simultaneously makes it possible to evaluate metabolic disorders with a minimal sample volume (10-50 µl of serum/plasma/medium from cultured cells). Panels that focus on endocrine, cardiovascular and inflammation areas of interest are available and can be customized to individual needs. Antibodies directed against targets of interest (cytokines, adipokines, cardiovascular markers) are coupled to internally dyed beads specific to each target. Thus, a minimal sample volume can yield information on multiple molecules of interest in a single assay.


Metabolite Analyses

The Core provides substrate determination of glucose, triglycerides and free fatty acids:

GLUCOSE: A commercially available kit based on glucose oxidase technology provides a quantitative enzymatic determination adapted to a microtiter plate format. The amount of serum required for this assay is 5 µl.

TRIGLYCERIDES: For tissue samples, lipids are extracted and content determined by enzymatic measurement of glycerol using a commercially available triglycerides kit. For serum or plasma, a “Triglyceride E” kit will be used. The assay is based on measurement of released glycerol by coupled enzyme reactions catalyzed by glycerol kinase, glycerol phosphate oxidase and peroxidase. Increase in absorbance is directly proportional to triglyceride concentration. Ten µl of serum or plasma is required per assay tube.

FREE FATTY ACIDS: Serum concentrations of FFA can be measured with diagnostic reagent kits using a microtiter plate assay according to kit specifications. Sufficient serum can be generated from 0.5-1.0 ml whole blood.


PI3K assay

In addition to identifying the regulatory and catalytic subunits of PI3K in tissue or cell lysates by Western blot, it is often of interest to measure the effects of stimulatory/inhibitory conditions on PI3K enzymatic activity. IRS-1-associated PI3K is immunoprecipated from prepared cell lysates then incubated with phospholipid substrate and radio-labeled ATP to form a radio-labeled product, which is isolated by TLC and visualized by autoradiography for quantitative analysis.


Pricing

Pricing lists for services are currently under develpment. Contact Rachel Janssen, lab manager, with questions (303-724-3979; rachel.janssen@ucdenver.edu).