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. Author manuscript; available in PMC: 2021 Jan 21.
Published in final edited form as: Anal Chem. 2020 Jan 7;92(2):1856–1864. doi: 10.1021/acs.analchem.9b03811

Figure 5.

Figure 5.

Application of TOXcms to identify off-target effects of siRNA. (a) Theoretical schematic showing expected results for a biochemical reaction in which substrate S is converted to product P by enzyme E. P is detected in wild-type (WT) samples, but not after knockdown (KD) of E. To ensure that P decreases due to enzyme knockdown, siRNA-resistant E is overexpressed in the KD samples to restore activity. Overexpression of E is expected to “rescue” levels of P. (b) In WT samples, acylcarntines are readily detected by LC/MS. After KD of CPT1, however, AC levels drop. AC levels are restored by expressing CPT1 that is resistant to siRNA. Data shown were output by TOXcms. (c) Example of a metabolic alteration identified by TOXcms to be a result of an off-target effect of siRNA, rather than an effect of CPT1 KD.