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
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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:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(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
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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:
Analyte Technology Platform DNA PCR DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(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