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Pre-storage centrifugation conditions have significant impact on measured microRNA levels in biobanked EDTA plasma samples.

Author(s): Binderup HG, Houlind K, Madsen JS, Brasen CL

Publication: Biochem Biophys Rep, 2016, Vol. 7, Page 195-200

PubMed ID: 28955906 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of different centrifugation protocols on platelet counts and miRNA levels. Additionally, the differences in platelet counts due to sampling location within the plasma phase of centrifuged blood was examined. Blood was collected from 10 healthy patients using a 21-gauge needle into three K2EDTA tubes. One tube from each patient was centrifuged at 3000 x g for 15 min and the resultant plasma was  then centrifuged at 3000 x g for 15 min to produce platelet-poor plasma (PPP) which was stored at -80°C. Another tube was centrifuged for 10 min at 2000 x g and the resultant plasma was frozen for a week at -80°C. Following thaw, the plasma was split and half was centrifuged at 3000 x g for 15 min to remove residual platelets, assayed, and then centrifuged again at 3000 x g for 15 min before reanalysis while the other half was analyzed, centrifuged at 3000 x g for 30 min, and then analyzed again. The third tube was centrifuged for 10 min at 2000 x g, the resultant plasma was transferred in 1 mL aliquots from the top down, and then each aliquot was analyzed separately. miRNA was isolated using the Nucleospin miRNA kit and reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit and Custom TaqMan MIR RT Pool and then quantified using custom TaqMan low density arrays. miRNA and cDNA were frozen at -20°C until use. Platelets were counted using a hematology autoanalyzer. 

   Summary of Findings:

   As expected, the highest platelet counts were observed in specimens subjected to a single centrifugation step (2000 x g for 10 min or 3000 x g for 15 min). Although residual platelets were observed in specimens subjected to centrifugation at 2000 x 10 min followed by centrifugation at 3000 x g for 15 min, the amount decreased to 0-1x10^9 (equivalent to PPP) for all specimens when centrifuged again for 15 min at 3000 x g or in 6 of 10 specimens when the second centrifugation was extended to 30 min.

   In specimens subjected to a single centrifugation for 10 min at 2000 x g, platelets were generally equally distributed throughout the tube but the lowest aliquot contained a higher number of platelets in two of the ten patients.

   Compared to PPP, plasma obtained by centrifugation at 2000 x g for 10 min with or without an additional centrifugation step after frozen storage had higher levels of miR-142-3p (4.7-37.3 fold, P<0.05), miR-145 (4.9-68.4 fold, P<0.05), miR-26a (2.4-92.3-fold, P<0.05), miR-28 (9.3-102.5-fold, P<0.05), miR-301 (11.8-84-fold, P<0.05), miR-30a-5p (2.5-9.8-fold, P<0.05), miR30-d (2.8-13.9-fold, P<0.05), miR-328 (23.0-94.1 fold, P<0.05), miR-331 (8.4-113.1-fold, P<0.05), miR-335 (17.1-95.1, P<0.05), miR-340 (8.3-53..7-fold, P<0.05), miR-92a (2.5-13.8 fold, P<0.05), and miR-93 (3.5-13.1 fold, P<0.05). With the exception of miR-26a, significant differences were observed in miR for all specimens centrifuged twice at 3000 x g for 15 min after frozen storage compared to PPP (P<0.001). When normalized to the exogenous control cel-miR-39, levels of miR-16 were found to be significantly higher in specimens stored at -80°C after an initial centrifugation at 2000 x g for 10 min than in PPP (1.6 to 2.6-fold, P<0.05, all). Consequently, levels of miR normalized to miR-16 showed smaller fold changes between protocols but most differences remained significant.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Hematology/ auto analyzer
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Biospecimen heterogeneity	Sampling at multiple levels
   Storage	Storage conditions	As PPP As plasma
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-16 miR-142-3p miR-145 miR-26a miR-28 miR-301 miR-30a-5p miR-30d miR-328 miR-331 miR-335 miR-340 miR-92a miR-93
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated Multiple durations compared Multiple speeds compared Different number of centrifugation steps compared

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