Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Stability of Circulating Blood-Based MicroRNAs - Pre-Analytic Methodological Considerations.

Author(s): Glinge C, Clauss S, Boddum K, Jabbari R, Jabbari J, Risgaard B, Tomsits P, Hildebrand B, Kääb S, Wakili R, Jespersen T, Tfelt-Hansen J

Publication: PLoS One, 2017, Vol. 12, Page e0167969

PubMed ID: 28151938 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of anticoagulant type, room temperature storage before or after centrifugation, shaking, frozen storage, and freeze-thaw cycling on levels of miR-1, miR-21, and miR-29b in plasma and serum. Blood was collected from 12 healthy volunteers at two collection locations (6 in Munich and 6 in Copenhagen) by venipuncture with a butterfly needle into serum separator tubes, EDTA tubes, lithium-heparin tubes, and citrate tubes (1 site only). Tubes were inverted 10-times after collection. Plasma and serum were obtained by centrifugation at 3000 rpm for 15 min (Copenhagen) or at 4000 rpm for 20 min (Munich). RNA was isolated from plasma and serum using the NucleoSpin1 miRNA plasma protocol. Real-time qPCR was conducted using TaqMan probes. The effect of room temperature storage was investigated by storing blood and plasma in EDTA tubes for 0, 4, 8, 12, 24, or 72 h (Copenhagen site only) and by storing blood and plasma/serum in EDTA or serum tubes for 0, 1, or 4 days (Munich site only). Effects of mechanical disturbance were investigated by storing blood, plasma, or serum on a shaker rotating at 30 rpm for 1 or 8 h, before or after centrifugation. Effects of frozen storage and freeze-thaw cycling were investigated by freezing the supernatant and storing at -80˚C for 1 day or 9 months and freeze-thawing once or four times. 

   Summary of Findings:

   Levels of miR-21 and miR-1 did not differ significantly among EDTA-plasma, serum and citrate-plasma, although neither were detectable in lithium heparin plasma. Room temperature storage of EDTA blood at the Copenhagen site for 72 h before centrifugation resulted in significant declines in miR-21 and miR-29b levels in EDTA-plasma (P<0.01, both). In the Munich cohort, storage of blood for 4 days at room temperature before centrifugation did not affect levels of miR-1 and miR-21 in EDTA-plasma or miR-1 levels in serum, but resulted in higher levels of miR-21 in serum. After processing, storage of EDTA-plasma at room temperature for 24 h or 4 days resulted in significant increases in levels of miR-21 (P<0.05 and P<0.01, respectively) and miR-1 (P<0.001 and P<0.01, respectively). Similarly, post-processing storage of serum for 24 h at room temperature increased miR-21 levels (P<0.01) while storage for 4 days increased levels of both miR-21 and miR-1 (P<0.001 and P<0.05, respectively). Physical disturbance of blood by storage on a shaker for 8 h pre-centrifugation did not affect miR-21 or miR-1 levels in EDTA-plasma, but resulted in an increase in miR-1 in serum (P<0.05). In contrast, when serum was stored on a shaker for 8 h post-processing miR-1 and miR-21 levels were not affected, but post-processing storage of EDTA plasma resulted in higher levels of miR-1 and miR-21  in EDTA-plasma (P<0.01, both). Although storing EDTA-plasma for 9 months at -80˚C did not alter miR-21 or miR-29b levels, storage of whole blood under identical conditions resulted in declines in miR-21 and miR-29b  (P<0.01 and P<0.05, respectively). Compared to specimens thawed once, those thawed four-times had significantly higher levels of miR-21 and miR-1 in EDTA-plasma (P<0.05, both) and higher levels of miR-21 in serum (P<0.01).

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen
   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Anticoagulant	EDTA Citrate Lithium heparin None
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum
   Biospecimen Aliquots and Components	Biospecimen mixing	Not shaken Shaken 1 h at 30 rpm Shaken 8 h at 30 rpm
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-1 miR-21 miR-29b
   Storage	Time at room temperature	0 h 4 h 8 h 12 h 24 h 72 h 1 day 4 days
   Storage	Storage duration	1 day 9 months
   Storage	Freeze/thaw cycling	0 cycles 4 cycles
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated

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2. Study Purpose

   This study compared levels of miR-21 and miR-29b among blood fractions. Blood was collected from three healthy volunteers by venipuncture with a butterfly needle into EDTA tubes. Tubes were inverted 10 times after collection. Blood fractions were obtained by centrifugation at 3000 rpm for 15 min or at 4000 rpm for 20 min, depending on collection site. RNA was isolated from plasma and serum using the NucleoSpin1 miRNA plasma protocol and real-time qPCR was conducted using TaqMan probes.

   Summary of Findings:

   Compared to plasma, miR-21 levels were significantly higher in buffy coat and red blood cells (P<0.01 and P<0.001, respectively) and miR-29b levels were significantly higher in buffy coat (P<0.01).

   Biospecimens

   • Bodily Fluid - Blood
   • Cell - Red Blood Cells
   • Bodily Fluid - Buffy Coat
   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Buffy coat Red blood cells

   View more