title: Centrifugation: an important pre-analytic procedure that influences plasma microRNA quantification during blood processing.
abstract: Circulating microRNAs are robustly present in plasma or serum and have become a research focus as biomarkers for tumor diagnosis and prognosis. Centrifugation is a necessary procedure for obtaining high-quality blood supernatant. Herein, we investigated one-step and two-step centrifugations, two centrifugal methods routinely used in microRNA study, to explore their effects on plasma microRNA quantification. The microRNAs obtained from one-step and two-step centrifugations were quantified by microarray and TaqMan-based real-time quantitative polymerase chain reaction (Q-PCR). Dynamic light scattering was performed to explore the difference underlying the two centrifugal methods. The results from the microarray containing 1,347 microRNAs showed that the signal detection rate was greatly decreased in the plasma sample prepared by two-step centrifugation. More importantly, the microRNAs missing in this plasma sample could be recovered and detected in the precipitate generated from the second centrifugation. Consistent with the results from microarray, a marked decrease of three representative microRNAs in two-step centrifugal plasma was validated by Q-PCR. According to the size distribution of all nanoparticles in plasma, there were fewer nanoparticles with size >1,000 nm in two-step centrifugal plasma. Our experiments directly demonstrated that different centrifugation methods produced distinct quantities of plasma microRNAs. Thus, exosomes or protein complexes containing microRNAs may be involved in large nanoparticle formation and may be precipitated after two-step centrifugation. Our results remind us that sample processing methods should be first considered in conducting research.
authors.affiliation: State Key Laboratory of Oncology in South China, Bank of Tumor Resource, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong 510060, P. R. China. jiaweih@mail.sysu.edu.cn.
authors.forename: Xiao-Hui
authors.initials: XH
authors.lastname: Zheng
authors.forename: Cui
authors.initials: C
authors.lastname: Cui
authors.forename: Xin-Xi
authors.initials: XX
authors.lastname: Zhou
authors.forename: Yi-Xin
authors.initials: YX
authors.lastname: Zeng
authors.forename: Wei-Hua
authors.initials: WH
authors.lastname: Jia
mesh_terms: D015415:Biomarkers / Q000097:blood; D002498:Centrifugation / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000097:blood*; D046228:Microarray Analysis; D053758:Nanoparticles; D010949:Plasma / Q000737:chemistry*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D015415:Biomarkers; D035683:MicroRNAs