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Published on: October 26, 2017
Centrifugation: an important pre-analytic procedure that influences plasma microRNA quantification during blood
Xiao-Hui Zheng1, Cui Cui, Xin-Xi Zhou
1State 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.
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.
Insights
Two-step centrifugation significantly reduces plasma microRNA detection by pelleting microRNAs within large nanoparticles. This impacts microRNA biomarker studies, necessitating careful consideration of sample processing methods for accurate quantification.
Area of Science:
- Biochemistry
- Molecular Biology
- Biomarker Discovery
Background:
- Circulating microRNAs (miRNAs) are valuable biomarkers for cancer diagnosis and prognosis.
- Plasma/serum miRNA quantification is crucial for biomarker studies.
- Centrifugation is a standard method for preparing plasma/serum samples.
Purpose of the Study:
- To compare the effects of one-step and two-step centrifugation on plasma microRNA quantification.
- To investigate the underlying mechanisms affecting miRNA yield based on centrifugation method.
Main Methods:
- Plasma samples were processed using one-step and two-step centrifugation.
- MicroRNA quantification was performed using microarray and quantitative real-time PCR (Q-PCR).
- Dynamic light scattering was used to analyze nanoparticle size distribution.
Main Results:
- Two-step centrifugation significantly decreased microRNA signal detection rates compared to one-step.
- Recoverable microRNAs were found in the precipitate from the second centrifugation step.
- A reduction in nanoparticles >1,000 nm was observed in two-step processed plasma.
- Q-PCR confirmed a marked decrease in three specific microRNAs after two-step centrifugation.
Conclusions:
- Centrifugation method critically affects plasma microRNA yield.
- Exosomes or protein complexes containing miRNAs may form large nanoparticles, leading to their loss during two-step centrifugation.
- Standardization of sample processing is essential for reliable miRNA biomarker research.
