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Updated: Apr 23, 2026
Isolation of Small Noncoding RNAs from Human Serum
Published on: June 19, 2014
Development of LC-MS methods for quantitation of hepcidin and demonstration of siRNA-mediated hepcidin suppression in
Siyang Li1, Tomoko Nakayama2, Akin Akinc2
1Barnett Institute, Northeastern University, Boston, MA 02115, United States.
Introduction:
A requisite step in developing a therapeutic to modulate the levels of hepcidin is the development of a quantitative method for measuring the concentration of serum hepcidin.
Methods:
To this end, an LC-MS method, based on selected reaction monitoring (SRM) with a triple quadrupole MS and an isotopically labeled hepcidin as internal standard, was developed to measure hepcidin in mouse and monkey sera.
Results:
Initially, 40 normal cynomolgus monkeys and 40 normal mice were studied to determine the normal endogenous levels of hepcidin, and an average of 50ng/mL was found in the monkeys and 46ng/mL in the mice. Next, experiments were conducted where an siRNA, targeting hepcidin, was administered to cynomolgus monkeys, resulting in effective hepcidin reduction (inhibition rate) of 87% after 24h and 74% after 48h, demonstrating to effectively reduce serume level of hepcidin.
Conclusions:
For better sensitivity, especially for the low volumes available for mouse sera, a second LC-MS method, based on parallel reaction monitoring (PRM) using a Orbitrap MS was developed and shown to be at least 10 fold lower in detection limits (or consumption of serum volume) than the SRM approach.
