Differential stability of cell-free circulating microRNAs: implications for their utilization as biomarkers.

Author(s): Köberle V, Pleli T, Schmithals C, Augusto Alonso E, Haupenthal J, Bönig H, Peveling-Oberhag J, Biondi RM, Zeuzem S, Kronenberger B, Waidmann O, Piiper A

Publication: PLoS One, 2013, Vol. 8, Page e75184

PubMed ID: 24073250 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of room temperature storage of serum, addition of RNase inhibitors to blood or serum, and blood cell lysis on miRNA levels in serum and in the vesicle and non-vesicle fractions of serum. Serum from healthy individuals was centrifuged for 30 min at 10,000 g to remove cell fragments, mixed with PBS and then centrifuged at 120,000 g for 2 h at 4 degrees C to separate vesicles from non vesicle fractions. miRNAs were extracted using the miRNeasy Mini Kit. The number of specimens was not specified for all experiments, but for those in which it was specified, 3-5 specimens were used.

   Summary of Findings:

   After 3 h at room temperature, levels of miR-1 and miR-122 were 40% of levels measured in fresh serum, but levels of miR-16, miR-21 and miR-142-3P remained at 75-80% of initial levels. After storage of serum for 24 h at room temperature, levels of miR-1 and miR-122 were 10% of the initial levels, levels of miR-16 and miR-21 were 40% of initial levels and levels of miR-142-3P were 60% of initial levels. The decreases in miR-16 and miR-122 levels were partially attenuated by the addition of 0.1 units/uL RNAse inhibitor to serum and almost completely prevented by the addition of 0.3 units/uL RNAse inhibitors to serum. Similarly, the levels of miR-122 were stabilized when RNAse inhibitors were added to blood prior to the isolation of serum. miR-122 levels were also stable in serum for 24 h at room temperature when blood was lysed with Triton-X but not when blood was left intact or RNAse A was added. The authors report that the addition of lysed red blood cells to serum also stabilized levels of miR-122. With increased incubation of serum, the ratio of miR-16, miR-122, miR-192 and miR-500 in the vesicle to the non-vesicle fraction of serum increased indicating faster degradation of supernatant miRNAs than vesicle-associated miRNAs. Further, levels of miR-16, miR-122 and miR-21 in the serum supernatant were more susceptible to RNAse A than those in the vesicles.

   Biospecimens

   • Bodily Fluid - Serum
   • Bodily Fluid - Blood

   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	Hemolysis	Absent Chemically-induced Hemolysate added
   Storage	Time at room temperature	0 h 1 h 3 h 5 h 24 h
   Analyte Extraction and Purification	RNase inactivation	RNAse inhibitor added to blood RNAse inhibitor added to serum No RNAse inhibitor added 0.1 units/uL RNAse inhibitor added 0.3 units/uL RNAse inhibitor added 0.5 units/uL RNAse inhibitor added
   Analyte Extraction and Purification	Nucleic acid digestion	RNAse A added No RNAse added
   Real-time qRT-PCR Specific	Technology platform	miR-1 miR-16 miR-21 miR-122 miR-142-3p miR-192 miR-500
   Biospecimen Aliquots and Components	Blood and blood products	Serum Serum supernatant Serum vesicles

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