Centrifugation: an important pre-analytic procedure that influences plasma microRNA quantification during blood processing.
Author(s): Zheng XH, Cui C, Zhou XX, Zeng YX, Jia WH
Publication: Chin J Cancer, 2013, Vol. 32, Page 667-72
PubMed ID: 23601242 PubMed Review Paper? No
Purpose of Paper
This paper compared microRNA (miR, miRNA) levels and particle sizes in plasma obtained from two EDTA blood specimens before and after a second centrifugation step.
Conclusion of Paper
More miRNAs were detected in plasma obtained from a single centrifugation (820 x g) than that obtained by the two-step centrifugation protocol which included a higher speed centrifugation step (16,000 x g). Further, levels of miR-BART5, miR-BART16, and U6 were lower in specimens centrifuged twice than those centrifuged once. The majority of miRNA lost during the second centrifugation step were detected in the precipitates. Nanoparticles detected in plasma centrifuged twice had a smaller mean size than that from plasma centrifuged once.
Studies
-
Study Purpose
This paper compared miRNA levels and particle sizes in plasma specimens obtained from two EDTA blood specimens before and after a second centrifugation step. Whole blood from two patients with nasopharyngeal carcinoma was collected in EDTA tubes and centrifuged at 820 x g for 10 min within 30 min of blood draw. A 1 mL aliquot from each specimen was transferred to a new tube and centrifuged at 16,000 x g for 10 min. The supernatant from the first and second centrifugations and the precipitates were stored at -80˚C. RNA was extracted from plasma using miRVana PARIS microRNA Isolation Kit and quantified by NanoDrop. Levels of two Epstein-Barr miRNAs (miR-BART5 and miR-BART16) and U6 were quantified by TaqMan Real-time PCR and miRNA profiling of 1347 miRNA was conducted using Agilent MicroArrays. Nanoparticle size distribution was investigated in undiluted plasma by dynamic light scattering.
Summary of Findings:
A total of 284 and 250 miRNAs were detected in the two plasma specimens obtained by a single centrifugation and 206 and 195 miRNAs were detected in plasma obtained by two-step centrifugation. Of these, 201 and 193 miRNAs were detected regardless of the number of centrifugation steps. The precipitates generated during the second centrifugation contained the majority of miRNA that were no longer found in plasma after the second centrifugation step (83 of 83 and 52 of 57). Specimens centrifuged twice had higher cycle threshold (CT) values than those centrifuged once for miR-BART5 (40 versus 33.5 and 37.4), miR-BART-16 (40 versus 33.5 and 35.6), and U6 (40 versus 34.8 and 33.8). Interestingly, light scatter analysis of nanoparticles revealed that the average size was smaller in plasma obtained by two centrifugation steps than in plasma obtained by a single centrifugation in both specimens (62.9 nm versus 169.9 nm and 28.6 nm versus 152 nm).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Cell count/volume Light scattering RNA DNA microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Different number of centrifugation steps compared