2 researchers in the laboratory examining results

Key Questions
 Considerations Before Contacting the Center for Services

Before contacting the Center for services, researchers should be prepared to discuss the following:

What is the scientific question or problem?

Examples:

  • I need to determine the change in molecule X when I do Y.
  • I want to know whether the oxidative branch of the pentose phosphate pathway is contributing to the production of NADPH.

What is the experimental system?

Cell culture, spheroids, animal models, human subject, PDX, etc.

Targeted or global metabolomics? Pathway analysis or flux analysis?

  • For these questions, often the researcher will not have an answer, but should expect that these aspects will be discussed.
  • It is highly recommended that the client be sure of the term flux, and refresh themselves on network dynamics and reaction diffusion systems under inhomogeneous conditions.

What information is already available?The consultation will then focus on the experimental design, which will also need to include biostatisticians for estimating sample sizes.

  • Transcriptomics data may imply metabolic consequences and may be partially validated by protein analysis (cf. Westerns, MS, enzyme activity).
  • The Seahorse XRF provides information about the rate of proton extrusion (ECAR) and the rate of oxygen consumption (OCR) by adherent cells under normoxic conditions.
    • By varying the extracellular nutrient source (e.g. glucose, glutamine, fatty acids) and with the use of specific inhibitors, it is possible to estimate net lactic fermentative flux, glycolytic reserve, respiration potential and the degree of coupling to oxidative phosphorylation. Such information is extremely valuable in designing metabolomics analyses or whether metabolism is a significant correlate of the biological function of interest.

Will the user do their own sample work up? What analytical techniques are needed? What level of data reduction is required?

The Center has SOPs. Questions that cannot be answered using these SOPs will be considered in terms of development time to establish specific metabolic assays.

  • This is relevant mainly in the context of a small number of metabolites (targeted metabolomics).

Costs

What are the costs associated with the samples being analyzed?

What are the costs of the metabolomics experiments, including tracers?

Data Analysis

It is VERY common for data analysis to take 90% of the metabolomics labor, so the researcher should be prepared to discuss the time frame and cost structure with this consideration, versus obtaining data analysis training for their own laboratory.

  • We have found, however, that the latter approach still incurs considerable labor for the CESB/RCSIRM staff, as data analysis is highly nuanced and extensive consulting is still typically needed.
  • Once an experimental approach has been worked out, the user will be introduced to laboratory personnel who will carry out the analyses and oversee the project progress- this person will then be the primary contact. Data collection is not the rate limiting step. Global analyses often produce hundreds to thousands of identifiable and quantifiable compounds or features but this means the reduction of raw data is extremely time consuming.