Extraction of DNA from serum for high-throughput genotyping: findings from pilot studies within the Prostate Cancer Prevention Trial.
OBJECTIVE
Deoxyribonucleic acid (DNA) extraction from blood and genotyping for candidate single nucleotide polymorphisms (SNP) is now an important part of almost all molecular epidemiologic studies. However, in many studies the amount of blood sample is limited or only serum is available. We conducted several pilot studies to identify methods for DNA extraction and high-throughput SNP genotyping of both white blood cell (WBC) and serum DNA that can be done centrally and reliably for large numbers of samples.


METHODS
We used biospecimens from the Prostate Cancer Prevention Trial (PCPT), a phase III, double-blind, placebo-controlled trial that tested the efficacy of finasteride for the primary prevention of prostate cancer. DNA was extracted from WBCs, from serum, and also from serum after organic solvent extraction for analysis of hormones. We also conducted blinded high-throughput genotyping in three laboratories to assess feasibility and reliability of results with differing methodologies using DNA from WBCs and from serum.


RESULTS
Genotyping of DNA extracted from WBCs resulted in highly reliable, reproducible results across laboratories using different genotyping platforms. However, genotyping with DNA extracted from serum did not provide reliable data using high-throughput multiplex approaches such as Sequenom (hME and iPLEX) and Applied Biosystems SNPlex, but was successful using Taqman.


CONCLUSIONS
Based on the results of these pilot studies, we conclude that DNA obtained from serum must be used judiciously, and that genotyping using multiplex methods is not suitable for serum DNA.
Hoque
Ashraful
A
University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
Goodman
Phyllis
P
Ambrosone
Christine B
CB
Figg
William D
WD
Price
Douglas K
DK
Kopp
William
W
Wu
Xifeng
X
Conroy
Jeffrey
J
Lehman
Teresa A
TA
Santella
Regina M
RM
D019963:Clinical Chemistry Tests / Q000379:methods; D004273:DNA, Neoplasm / Q000032:analysis* / Q000097:blood; D005838:Genotype; D006801:Humans; D007962:Leukocytes / Q000737:chemistry*; D008297:Male; D010865:Pilot Projects; D011471:Prostatic Neoplasms / Q000097:blood / Q000235:genetics*
D004273:DNA, Neoplasm