The effect of centrifugation on circulating mRNA quantitation opens up a new scenario in expression profiling from patients with metastatic colorectal cancer

Objectives: To investigate the effect of centrifugation in circulating mRNA quantitation and to design a new, consistent protocol for it. Design and methods: The concentrations of β-catenin mRNA, P-selectin mRNA, cytokeratin 20 mRNA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the plasma, the mononuclear and red cells (MARC) from metastatic colorectal (CRC) patients whose blood samples were centrifuged with 2 different forces, were measured. A new protocol to measure expression profiles in MARC was studied. Results: When the centrifugal force was increased: (1) the concentrations of all transcripts were dramatically decreased in the plasma samples; (2) only the concentrations of P-selectin mRNA and GAPDH mRNA were significantly increased in the mononuclear cell samples; and (3) their concentrations in the red cells did not change significantly. The new protocol found 9 differential expressed genes. Conclusions: Centrifugal force causes inconsistent mRNA quantitation in the plasma. The new protocol discovered 9 differential expressed genes for metastatic CRC.