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

Isolation and extraction of circulating tumor DNA from patients with small cell lung cancer.

Author(s): Board RE, Williams VS, Knight L, Shaw J, Greystoke A, Ranson M, Dive C, Blackhall FH, Hughes A

Publication: Ann N Y Acad Sci, 2008, Vol. 1137, Page 98-107

PubMed ID: 18837931 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine the effects of extraction method, delayed centrifugation, and diagnosis on DNA yield and fragment sizes from plasma and serum.

Conclusion of Paper

DNA yield was highest when extracted using the QIAamp Viral spin kit, when serum was used rather than plasma and when serum was allowed to clot for 24 h rather than 30-60 min. In contrast to serum, DNA yield in plasma was not affected by delayed centrifugation. Compared with healthy controls, patients with small cell lung cancer (SCLC) had higher levels of DNA extracted from plasma but not serum, a higher success rate for amplification of a 512 bp fragment of Bcl-2 from plasma and serum, and a higher ratio of the 272 bp fragment of p56 to the 77 bp fragment of AAT in serum and plasma.

Studies

  1. Study Purpose

    The purpose of this study was to determine the effect of serum clotting time and DNA extraction kit on DNA yield from serum and plasma from 5 healthy volunteers. For serum, blood was left to clot at room temperature for 30-60 min or 24 h, while plasma was obtained by immediate centrifugation. Serum and plasma were stored at -80°C until DNA isolation, and DNA was stored at -20°C. DNA yield was determined by real-time PCR amplification of α1-antitrypsin (AAT).

    Summary of Findings:

    DNA yield from plasma and serum (both clot times) was highest using the QIAamp Viral spin kit followed by the Agencourt kit which yielded only 25-35% as much DNA. Importantly, regardless of kit choice, the DNA yield was higher from serum than plasma and increased when serum was allowed to clot for 24 h rather than 30-60 min before centrifugation. Using the QIAamp Viral spin kit, the DNA yield was ~5 fold higher from serum with a 30-60 min clot time than from plasma. When the clot time was increased to 24 h from 30-60 min, the DNA yield from serum doubled, resulting in ~10 fold higher levels of DNA extracted from serum than from plasma.

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    DNA Real-time qPCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Analyte Extraction and Purification Analyte isolation method QIAamp DNA Mini Blood kit
    Agencourt Genfind Blood and Serum Genomic DNA Isolation kit
    QIAamp Virus Spin kit
    Invitrogen ChargeSwitch gDNA Serum kit
    Real-time qPCR Specific Targeted nucleic acid AAT
    Biospecimen Aliquots and Components Blood and blood products Plasma
    Serum
    Storage Time at room temperature 30-60 min
    24 h
  2. Study Purpose

    The purpose of this study was to determine the effect of delayed centrifugation, using serum rather than plasma and diagnosis on DNA yield, as determined by real-time PCR, and on PCR fragment sizes. Blood from 10 healthy volunteers and 10 patients with SCLC was collected into EDTA and serum gel Vacutainer tubes. To investigate the effects of delayed centrifugation on plasma, only specimens from healthy individuals were used. Both serum and plasma were stored frozen in fresh tubes at -80°C. DNA was extracted using the QIAamp virus spin kit and stored at -20°C.

    Summary of Findings:

    DNA yield from plasma was not affected by delayed centrifugation of blood, but yields increased ~3 fold in serum when blood clot time was increased from 30-60 min to 24 h, regardless of diagnosis. Plasma DNA levels were higher in patients with SCLC than in healthy controls (15.6 ng/mL versus 5.07 ng/mL, p=0.002), but diagnosis had no effect on serum DNA levels. The 512 bp fragment of Bcl-2 was amplifiable in all plasma and serum from patients with SCLC but only in 4/10 serum and 0/10 plasma specimens from healthy controls. Patients with SCLC had a higher ratio of the 272 bp fragment of p56 to the 77 bp fragment of AAT than healthy individuals in serum with 30-60 min clot time (44% versus 24%, p=0.05), serum with 24 h clot time (47% versus 20%, p=0.05) and plasma (13% versus 8%, p=0.04).

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Neoplastic - Carcinoma
    • Normal
    Platform:
    AnalyteTechnology Platform
    DNA PCR
    DNA Real-time qPCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Preaquisition Diagnosis/ patient condition SCLC
    Healthy
    Biospecimen Aliquots and Components Blood and blood products Plasma
    Serum
    Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
    Real-time qPCR Specific Targeted nucleic acid AAT
    PCR Specific Targeted nucleic acid AAT
    p53
    Bcl-2
    PCR Specific Length of gene fragment 77 bp
    272 bp
    512 bp
    Storage Time at room temperature <30 min
    30-60 min
    2 h
    4 h
    8 h
    24 h

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