Use of magnetic beads for plasma cell-free DNA extraction: toward automation of plasma DNA analysis for molecular diagnostics.
Author(s): Stemmer C, Beau-Faller M, Pencreac'h E, Guerin E, Schneider A, Jaqmin D, Quoix E, Gaub MP, Oudet P
Publication: Clin Chem, 2003, Vol. 49, Page 1953-5
PubMed ID: 14578335 PubMed Review Paper? No
Purpose of Paper
This paper investigated if the automated Kingfisher DNA extraction kit was comparable to the Qiagen kit in yield of cell-free DNA and investigated the impact of plasma concentration and proteinase K digestion on DNA yield.
Conclusion of Paper
The automated Kingfisher kit was comparable to the Qiagen kit in DNA yields from concentrated plasma but resulted in lower yields from non-concentrated plasma. Importantly, the DNA obtained by the automated method was of suitable quality for real-time PCR and microsatellite analysis.
Studies
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Study Purpose
This study investigated if the automated Kingfisher DNA extraction kit was comparable to the Qiagen kit in yield and quality of cell-free DNA and investigated the impact of plasma concentration and proteinase K digestion on DNA yield. Two EDTA blood specimens were obtained from each of 19 patients with lung cancer, 4 patients with colon cancer, and 20 healthy patients. Plasma was obtained by centrifugation at 800 x g for 10 min and then recentrifuged for 10 min at 1500 x g in a polypropylene tube. Proteins were digested by 1 h incubation with proteinase K at 37˚C in a Tris/SDS/EDTA buffer. Digests were concentrated and filtered by centrifugation at 2600 x g for 20 min in Amicon Ultra-15 filtration devices. The concentrates were stored at -20˚C until extraction. DNA was extracted using KingFisher silicate magnetic beads and a KingFisher ML robotic magnetic particle processor or using the Qiagen QIAamp DNA Midi reagent.
Summary of Findings:
The authors report that protein digestion with proteinase K was necessary to obtain DNA. While DNA yield from the Qiagen kit was not significantly affected by plasma concentration, the yield obtained with KingFisher isolation from concentrated plasma was higher than that obtained with the Qiagen kit from unconcentrated plasma (P=0.009) but was comparable from concentrated plasma (P=0.421). Importantly, the DNA obtained using the automated KingFisher method was of suitable quality for microsatellite amplification, DNA sequencing, and real-time PCR. Although DNA yields were comparable between plasma from cancer patients and healthy controls, the cycle threshold values for GAPDH were higher in specimens from patients than healthy controls.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Cancer
Healthy
Biospecimen Aliquots and Components Blood processing method Plasma concentrated
Plasma not concentrated
Analyte Extraction and Purification Analyte isolation method KingFisher kit
Qiagen kit
Analyte Extraction and Purification Protein digestion Proteinase K
No digestion
Real-time qPCR Specific Targeted nucleic acid GAPDH