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

The Effects of Different Storage Conditions and Repeated Freeze/Thaw Cycles on the Concentration, Purity and Integrity of Genomic DNA.

Author(s): Safarikova M, Kubena AA, Frankova V, Zima T, Kalousova M

Publication: Folia Biol (Praha), 2021, Vol. 67, Page 10-15

PubMed ID: 34273262 PubMed Review Paper? No

Purpose of Paper

This paper compared the concentration and purity of DNA isolated from EDTA plasma using the MagNA Pure Kit or a salting-out based method; DNA samples were also freeze-thaw cycled between 0 and 100 times at -20°C, -80°C, or in liquid nitrogen.

Conclusion of Paper

DNA yield was higher when extraction from EDTA plasma was with the salting-out method than the MagNA Pure Kit. Analysis of variance (ANOVA) revealed an effect of freeze-thaw cycling on DNA concentration and an interaction between cycle number and freezing temperature. However, mixed model analysis indicated that significant effects of freeze-thaw cycling were limited to DNA isolated using the MagNA Pure Kit and only when DNA was frozen in liquid nitrogen. In pairwise comparisons, the concentration of DNA was unaffected by freeze-thaw cycling at -20°C regardless of extraction method or by freeze-thaw cycling of DNA extracted by the salting-out method when DNA was stored at -80°C. A single freeze-thaw cycle affected DNA concentration when DNA was frozen in liquid nitrogen, regardless of extraction method. There was no effect of freeze-thaw cycling on DNA purity as assessed by the ratio of absorbance at 260 to 280 nm.

Studies

  1. Study Purpose

    This study compared the concentration and purity of DNA isolated from EDTA plasma using the MagNA Pure Kit or a salting-out based method; DNA samples were also freeze-thaw cycled between 0 and 100 times at -20°C, -80°C, or in liquid nitrogen. EDTA blood was collected from 10 healthy volunteers (5 men and 5 women) and centrifuged at 3,000 rpm for 15 min. The collected cells were stored at 2-8°C (duration not specified) before DNA was extracted by the salting out method and dilution in Tris EDTA, pH 7.6 or the MagNA Pure Compact Nucleic Acid Isolation Kit I on a MagNA Pure Compact System. DNA was stored at 2-8°C until the freeze-thaw cycling experiment. Matched aliquots of DNA were stored at 4°C or frozen at -20°C, -80°C, or in liquid nitrogen for 3 h followed by thawing at room temperature for 30 min; DNA samples were analyzed after 1, 2, 3, 4, 5, 10, 20, 50, and 100 cycles. DNA was quantified by spectrophotometer and DNA integrity was assessed by gel electrophoresis.

    Summary of Findings:

    DNA yield was higher when extraction from EDTA plasma was with the salting-out method than the MagNA Pure Kit (270.6±25.1 ng/µL versus 125±21.4 ng/µL, P<0.01). ANOVA revealed an effect of freeze-thaw cycling on DNA concentration (P=0.022) and an interaction between cycle number and freezing temperature (P<0.001). However, in the mixed model, only DNA that was isolated using the MagNA Pure Kit was significantly affected by freeze-thaw cycling and only when the isolated DNA was frozen in liquid nitrogen (P = 0.009). In pairwise comparisons, the concentration of DNA was unaffected by freeze-thaw cycling at -20°C, regardless of extraction method. Although freeze-thaw cycling at -80°C did not affect the concentration of DNA that was isolated by the salting-out method, DNA concentration was 10% higher after one cycle than 0 cycles (P<0.01) and 5.1% lower after 50 cycles than 1 cycle (P<0.01) when DNA was extracted using the MagNA Pure Kit. A single freeze-thaw cycle in liquid nitrogen led to a 15.7% and 40.2% increase in DNA concentration when DNA was isolated by the salting-out method and with the MagNA Pure Kit, respectively (P<0.01, both). The concentration of DNA that underwent freeze-thaw cycling in liquid nitrogen ≥5 times was lower than when DNA was freeze-thaw cycled in liquid nitrogen once, regardless of extraction method. There was no effect of freeze-thaw cycling on DNA purity as assessed by the ratio of absorbance at 260 to 280 nm.

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    DNA Electrophoresis
    DNA Spectrophotometry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Analyte Extraction and Purification Analyte isolation method Salting out
    MagNA Pure
    Biospecimen Preservation Cooling or freezing method/ rate At -20 degrees C
    At -80 degrees C
    Liquid nitrogen
    Storage Freeze/thaw cycling 0 cycles
    1 cycle
    2 cycles
    3 cycles
    4 cycles
    5 cycles
    10 cycles
    20 cycles
    50 cycles
    100 cycles

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