Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Comparison of the Effectiveness of Four Commercial DNA Extraction Kits on Fresh and Frozen Human Milk Samples.

Author(s): Butler C, Matsumoto A, Rutherford C, Lima HK

Publication: Methods Protoc, 2022, Vol. 5, Page

PubMed ID: 35893589 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this paper was to compare the yield and purity of DNA isolated from fresh and frozen aliquots of a single breast milk specimen following DNA extraction with four different kits. Breast milk from a single donor was aliquoted and processed immediately or stored frozen at -80°C for 3 weeks before DNA extraction. DNA was extracted from 200 µL of case-matched fresh and frozen milk aliquots using the Omega Biotek EZNA Blood DNA Mini Kit, NuceloSpin Food Mini Kit, and the Norgen Plasma/Serum Circulating DNA Purification Kit. Additionally, DNA was extracted from fresh specimens using the Norgen Milk DNA Preservation and Isolation Kit and then stored for 3 weeks at room temperature after the preservation step. DNA yield and purity (absorbance at 260/280 nm and 260/230 nm) were analyzed using a NanoDrop spectrometer.

   Summary of Findings:

   The ratio of absorbance (A) at 260 nm to 230 nm (A260/A230) was significantly lower when DNA was extracted using the Milk DNA Preservation and Isolation Kit than any of the other kits examined (P<0.05, for all), indicating a high degree of carbohydrate contamination. Further, DNA isolated with the Milk DNA Preservation and Isolation Kit also had the lowest A260/A80 ratios, indicating the presence of proteins or other contaminants.  DNA isolated from breast milk using the NuceloSpin Food Mini Kit led to the highest average A260/A230 ratio from fresh aliquots and the highest average A260/280 ratio from frozen aliquots, although differences were not consistent among aliquots as difference were not significant and only observed in either fresh or frozen aliquots (not both).  Variation in A260/A230 and A260/A280 ratios were lowest when DNA extraction from fresh or frozen breast milk was with the EZNA Blood DNA Mini Kit or the Plasma/Serum Circulating DNA Purification Kit, although the average A260/A280 ratio was slightly higher when the Plasma/Serum Circulating DNA Purification Mini Kit was used compared to the EZNA Blood DNA Mini Kit. Importantly, no differences were found between fresh and frozen aliquots when extraction was with the Plasma/Serum Circulating DNA Purification Mini Kit or the EZNA Blood DNA Mini Kit.

   Mean DNA yield was highest when DNA was extracted from fresh or frozen breast milk aliquots using the Plasma/Serum Circulating DNA Purification Mini Kit followed by the EZNA Blood DNA Mini Kit, the Milk DNA Preservation and Isolation Kit, and the NuceloSpin Food Mini Kit, regardless of whether aliquots were fresh or frozen.

    

   Biospecimens

   • Bodily Fluid - Breast Milk

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	None (fresh) Frozen
   Analyte Extraction and Purification	Analyte isolation method	Omega Biotek EZNA Blood DNA Mini Kit NuceloSpin Food Mini Kit Norgen Plasma/Serum Circulating DNA Purification Kit Norgen Milk DNA Preservation and Isolation Kit
   Storage	Storage duration	0 days 3 weeks

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