NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

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

The purpose of this paper was to determine the effects of additional centrifugation of plasma or serum, storage of serum and contamination of serum with hemolysate on quantification of microRNAs (miR) in healthy individuals.

Conclusion of Paper

Of the five column-based miRNA extraction kits evaluated, Trizol-LS produced miRNA samples with the lowest variability and cycle threshold (CT) values when analyzed immediately or after 6 months of storage at -20 degrees C. Significantly higher levels of miRNA-15b, miR-16, and miR-24 were found in plasma than in serum. A second centrifugation at 15,000 x g significantly reduced miR-15b, miR-16 and miR-24 levels in plasma and miR-24 in serum. A third centrifugation at 355,000 x g resulted in a modest albeit significant decline in miR-15b levels in both plasma and serum. Serum levels of miR-16 and miR-24 decreased after 24-72 h of storage post-extraction when compared to 0 h controls, regardless of temperature. Levels of miR-15b, which displayed a high level of interassay variability, were also affected by 24-72 h of storage post-extraction at all temperatures examined (ambient, 4 and -20 degrees C) although temperature- and duration-dependent trends were not apparent. Contamination of serum with hemolysate resulted in a concentration-dependent and progressive increase in miR-15b, miR-16 and miR-24 levels, although miR-122 levels were unaffected.

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
    Preservative Types
    • None (Fresh)
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    RNA Real-time qRT-PCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(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
  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
    Preservative Types
    • None (Fresh)
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    RNA Real-time qRT-PCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(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
  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
    Preservative Types
    • Frozen
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    RNA Real-time qRT-PCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(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
  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
    Preservative Types
    • None (Fresh)
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    RNA Real-time qRT-PCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(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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