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Quantification of microRNA levels in plasma - Impact of preanalytical and analytical conditions.

Author(s): Binderup HG, Madsen JS, Heegaard NHH, Houlind K, Andersen RF, Brasen CL

Publication: PLoS One, 2018, Vol. 13, Page e0201069

PubMed ID: 30024941 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of draw order, centrifugation steps, and normalization method on the measurement of miRNA levels in plasma from a single patient and compared levels of miRNA in standard plasma and PPP. Blood was collected using 21-gauge needles into K₂EDTA tubes with intermittent claudication after discarding the first 3 mL. PPP was obtained from 30 blood specimens from a single healthy patient using a standard dual centrifugation protocol (two steps at 3000 x g for 15 min) and by a prolonged single centrifugation (3000 x g for 30 min). Blood from 50 healthy patients was used to obtain standard plasma by centrifugation at 2000 x g for 10 min and PPP by the standard dual centrifugation protocol. Plasma was thawed and recentrifuged at 3000 x g for 15 min and miRNA was purified using the NucleoSpin miRNA Plasma kit. Levels of miR-92a, miR-126, and miR-16 were quantified by TaqMan real-time PCR assays, after preamplification using the TaqMan PreAmp Master kit using TaqMan low density arrays, and by ddPCR.

   Summary of Findings:

   Draw order had no effect on the levels miRNA in specimens from a single patient, regardless of normalization method. Levels of miR-92a, miR-126, and miR-16 were comparable in plasma obtained by dual centrifugation and prolonged single centrifugation when normalized to cel-miR-39, but levels of miR126 were higher in plasma obtained by dual centrifugation than that obtained by a single prolonged centrifugation step when normalized to miR-16 (P<0.02). The coefficients of variation depended on the normalization method and was highest when normalized to cel-miR-39 (10.9-21.1%) and lower when normalized to miR-16 alone (9.5-14.2%) or in combination with cel-miR-39 (9.2-13.3%). Centrifugation protocol was found to account for 64-73% of the intra-assay variability when normalized to cel-miR-39, 39-66% when normalized to miR-16, and 55-61% when normalized to both cel-miR-39 and miR-16.

   Cel-miR-39 normalized miRNA levels in PPP were not correlated to those in standard plasma, but miR-92a and miR-126 levels in PPP were modestly correlated to those in standard plasma when normalized to miR-16 (ρ=0.42, P=0.0024 and ρ=0.48, P=0.0004, respectively). Further, levels of miR-126 were modestly correlated in standard and PPP when normalized to cel-miR-39 in combination with miR-16, but there was no correlation observed for miR-92a levels normalized to cel-miR-39 with miR-16.

   Levels of miR-92a were not correlated between individual TaqMan assays and TLDA assays, but modest correlations between methods were observed for miR-16 (ρ=0.47, P=0.0005). Inter-assay precision was slightly higher for qPCR than ddPCR, but intra-assay precision was significantly higher with ddPCR.  miRNA levels were very strongly correlated between qPCR and ddPCR when normalized to cel-miR-39 (ρ≥0.92, P<0.0001), but only modest to very strong when normalized to miR-16 alone or in combination with cel-miR-39 (ρ=0.57-0.94, P<0.0001).

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Low density array
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Aliquot sequential collection	1st collection 2nd collection 3rd collection 4th collection 5th collection 6th collection 7th collection 8th collection 9th collection 10th collection
   Real-time qRT-PCR Specific	Data handling	Normalized to cel-miR-39 Normalized to miR-16 Normalized to cel-miR-39 and miR-16
   Real-time qRT-PCR Specific	Technology platform	ddPCR Single TaqMan assay TLDA
   Biospecimen Aliquots and Components	Centrifugation	Different number of centrifugation steps compared
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Platelet-poor plasma

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