image   How IROA Works Video

The IROA workflow schematic is presented below. A homogenous cell population is divided into two populations, a “Control” population and an “Experimental” population which will be subsequently treated with vehicle, or a stressor, respectively. All of the biological compounds in the control sample are labeled with “C13 media” signatures and all of the biological compounds in the experimental sample are labeled with “C12 media” signatures. The key element is that both the C12 and C13 media are chemically identical but isotopically different. Unlike other techniques, all of the carbon sources in the C12 media are randomly and universally composed of approximately 95% 12C with 5% 13C. Conversely, all of the carbon sources in the C13 media are composed of 95% 13C with 5% 12C, and again importantly, the isotopes are universally and randomly incorporated into all carbon positions.

The Control and the Experimental cell samples are grown in the IROA isotopically-defined media for a sufficient time to replace their original natural abundance carbon so that all of their contents will demonstrate distinctive isotopic patterns (orange box). Once labeled, the Experimental sample is treated with a stressing regimen, and the Control sample is treated with only vehicle for the same period of time. The stressing agent may be chemical, genetic, environmental, or any element or combination of elements that induce physiological alteration. At the conclusion of the experiment the experimental sample is mixed with a control sample and the pooled sample prepped and analyzed in a single analysis (green box). This has the effect of reducing sample-to-sample variance, and increasing data quality. For a discussion on data analysis, see “the ClusterFinderTM Software” section.

The Basic IROA® method grows isotopically labeled cells which are then treated experimentally. When pooled cells are processed the signals for the compounds from both the Control and Experimental cells may be distinguished and differences between the ratio of their areas are directly indicative of the ratio of the respective sizes of their metabolic pools. Outliers to the normalized ratios are metabolic pools that are impacted by the experimental treatment.