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.

Figure 1.. Signal detection rate of microRNAs.

A, the one-step (left) and two-step (middle) centrifugation plasma preparations and the precipitate (right) generated after the second centrifugation were obtained from each patient. B, according to the positive signals on the microarray, the signal detection rate of microRNAs was calculated. There is a marked decrease in signal detection rate after the second centrifugation in both samples.

Figure 2.. Ct values of representative microRNAs.

The Ct values of miR-BART5 (A), miR-BART16 (B), and U6 snRNA (C) in positive control (NPC tissue samples), negative control (blank), and plasma from one-step and two-step centrifugations were determined by quantitative polymerase chain reaction (Q-PCR). The content of representative microRNAs was greatly decreased after two-step centrifugation.

Figure 3.. Size distribution of nanoparticles in different plasma preparations.

A, size distribution of nanoparticles in plasma from one-step and two-step centrifugations in sample 1. B, size distribution of nanoparticles in plasma from one-step and two-step centrifugations in sample 2. C, the overall mean diameter of all nanoparticles in sample 1 and sample 2.