Analysis of circulating microRNA: preanalytical and analytical challenges.

Author(s): McDonald JS, Milosevic D, Reddi HV, Grebe SK, Algeciras-Schimnich A

Publication: Clin Chem, 2011, Vol. 57, Page 833-40

PubMed ID: 21487102 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 additional centrifugation steps on the levels of miR-15b, miR-16 and miR-24 in plasma and serum from 10 healthy volunteers. An initial centrifugation at 795 x g for 20 min was used to obtain the serum and plasma. Additional centrifugation of plasma and serum first at 15,000 x g for 10 min at an unspecified temperature and then at 355,000 x g for 1 h at 4 degrees C were performed on aliquots.

   Summary of Findings:

   Significantly higher levels of miR-15b (p=0.002), miR-16 (p=0.02), and miR-24 (p=0.002) were found in plasma than in serum. After a second centrifugation at 15000 x g for 10 min miR-15b, miR-24 and miR-16 were reduced in plasma (p=0.004, p=0.002, and p=0.02 respectively) and to a lesser extent in serum (p=0.02, p=0.002 and p=0.92, respectively). A third centrifugation at 355,000 x g for 1 h, had no effect on miR-24 and miR-17 levels, but slightly decreased miR-15 levels in plasma (p=0.008) and serum (p=0.002).

   Biospecimens

   • Bodily Fluid - Serum
   • 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 Serum
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-15b miR-16 miR-24
   Biospecimen Aliquots and Components	Centrifugation	Different number of centrifugation steps compared Multiple speeds compared

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2. Study Purpose

   The purpose of this study was to determine the effects of storage duration and temperature on the levels of miR-15b, miR-16 and miR-24 in serum from 10 healthy volunteers. miR extraction was performed on different days.

   Summary of Findings:

   Generally, levels of miR-15b, miR-16 and miR-24 decreased with storage. Levels of miR-16 and miR-24 decreased progressively with increasing storage at room temperature (p<0.001, both), increased in specimens stored for 24 h at 4 or -20 degrees C, and decreased in specimens stored for 48 h or 72 h at 4 or -20 degrees C. The magnitude of change in miR-16 and miR-24 levels was largest in specimens stored at room temperature, and was slightly smaller in specimens stored at 4 degrees C compared to those stored at -20 degrees C. Levels of miR-15b fluctuated after 24-72 h of storage at all temperatures examined although no trends with regard to temperature or duration were apparent. Quantification of miR-15b showed the highest interassay variability.

   Biospecimens

   • Bodily Fluid - Serum

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-15b miR-16 miR-24
   Storage	Storage temperature	-20 degrees C 4 degrees C Room temperature
   Storage	Storage duration	0 h 24 h 48 h 72 h

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3. Study Purpose

   The purpose of this study was to determine the effects of contamination with varying concentrations of hemolysate on the levels of miR-15b, miR-16, miR-24, and miR-122 in serum from 10 healthy volunteers. Hemolysate was produced by freezing red blood cells and was then added to aliquots of frozen serum from the same individual.

   Summary of Findings:

   Levels of miR-15b and miR-16 progressively increased with hemolysate by 2-4 fold, with significant effects observed with as little as 25 mg/dL hemoglobin. Levels of miR-24 exhibited similar increases in magnitude following the addition of 600 mg/dL or more of hemoglobin. In contrast, there was no effect of hemolysate on quantification of miR-122 or of added C. elegans miR-39 (cel- miR-39).

   Biospecimens

   • Bodily Fluid - Serum

   Preservative Types

   • Frozen

   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	Freeze/thaw-induced Hemolysate added No hemolysate added
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-16 miR-24 miR-15b miR-122 cel-miR-39
   Biospecimen Aliquots and Components	Biospecimen components	0 mg/dL Hemoglobin 25 mg/dL Hemoglobin 50 mg/dL Hemoglobin 75 mg/dL Hemoglobin 150 mg/dL Hemoglobin 300 mg/dL Hemoglobin 600 mg/dL Hemoglobin 1200 mg/dL Hemoglobin

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4. Study Purpose

   The purpose of this study was to determine which miRNA extraction kit produced the greatest yield of amplifiable miRNA and the least variability. miRNA was extracted from serum collected from 4 healthy individuals. miRNA extracted via the mirVana PARIS, miRNeasy Mini Kit, mirPremier microRNA Isolation Kit, and High Pure miRNA Isolation Kit was quantified immediately for the majority of specimens, although a subset of samples isolated with the mirVana PARIS and most specimens isolated using Trizol-LS were stored for 6 months at -20 degrees C prior to quantification.

   Summary of Findings:

   Among the column-based kits, the lowest cycle threshold (CT) values for miR-15b and miR-16 were obtained using the mirVana PARIS kit, although slightly lower CT values for miR-24 were obtained in specimens extracted using the miRNeasy Mini Kit compared to the mirVana PARIS kit (28.79 versus 28.87). Among column-based kits, variance in miR-15b and miR-16 CT values were lowest when the miRNeasy Mini Kit was used, while extraction with the mirVana PARIS kit resulted in the lowest variance in miR-24 CT values. However, miRNA isolated using Trizol-LS exhibited lower CT values and less CT variance in comparison to the mirVana PARIS kit when extracted samples were stored for 6 months at -20 degrees C prior to quantification. While not directly compared, CT values and variance were higher in samples stored for 6 months than those analyzed immediately after isolation.

   Biospecimens

   • Bodily Fluid - Serum

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	mirVana PARIS miRNeasy Mini Kit mirPremier microRNA Isolation Kit High Pure miRNA Isolation Kit Trizol-LS
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-15b miR-16 miR-24
   Storage	Storage duration	0 months 6 months

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