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.

The validity of circulating microRNAs in oncology: Five years of challenges and contradictions

“…A reduction of miRNA yield as a result of centrifugation of plasma has been reported in other studies (e.g. Page et al., 2013; Zheng et al., 2013). Important to note is that centrifugation of serum or plasma did not affect the recovery efficiency of spiked‐in synthetic Caenorhabditis elegans miRNA (cel‐miR‐54) (McDonald et al., 2011).…”

“…Regardless of the type of nucleic acid isolation, centrifugation has a strong impact on miRNA recovery. It was recently demonstrated that a two‐step centrifugation process during blood phase separation reduces the number of miRNAs collected from plasma compared to a one‐step centrifugation, and the authors hypothesized that miRNA‐containing particles may clump together and precipitate at high centrifugal speeds (Zheng et al., 2013). Furthermore, the effect of centrifugation as a preclearance step is sample type and miRNA dependent.…”

Circulating microRNA biomarkers for lung cancer detection in Western populations

“…Sample preparation is an essential pre‐analytical factor affecting the identification of potential marker candidates. Since the concentrations of cellular miRNAs are relatively high compared to those in plasma and serum, a second high‐speed centrifugation or filtration step during blood processing is recommended . This would serve to remove the potentially retained cells and cell debris from the plasma or serum fraction to minimize the possibility of blood cell contamination of the samples which could lead to an erroneous interpretation of the results.…”

“…Since the concentrations of cellular miRNAs are relatively high compared to those in plasma and serum, a second highspeed centrifugation or filtration step during blood processing is recommended. [40][41][42][43] This would serve to remove the potentially retained cells and cell debris from the plasma or serum fraction to minimize the possibility of blood cell contamination of the samples which could lead to an erroneous interpretation of the results. However, only few of the included studies applied such a high-speed centrifugation step (Table S4).…”

Analysis of circulating microRNAs in adrenocortical tumors

“…35 We used one-step centrifugation since this is the routine laboratory practice for plasma samples and Zheng et al showed that two-step centrifugation resulted in loss of 10-20% of all miRNAs present in plasma. These data together suggest that standardization of sampling is required for accurate miRNA measurements.…”