Quantitation of DNA in buccal cell samples collected in epidemiological studies.
Buccal cell samples are increasingly used in epidemiological studies as a source of genomic DNA. The accurate and precise quantitation of human DNA is critical for the optimal use of these samples. However, it is complicated by the presence of bacterial DNA and wide inter-individual variation in DNA concentration from buccal cell collections. The paper evaluated the use of ultraviolet light (UV) spectroscopy, Höechst (H33258) and PicoGreen as measures of total DNA, and real-time quantitative polymerase chain reaction (PCR) as a measure of human amplifiable DNA in buccal samples. Using serially diluted white blood cell DNA samples (at a concentration range of 300 to 0.5 ng microl-1), UV spectroscopy showed the largest bias, followed by Höechst, especially for low concentrations. PicoGreen and real-time PCR provided the most accurate and precise estimates across the range of concentrations evaluated, although an increase in bias with decreasing concentrations was observed. The ratio of real-time PCR to PicoGreen provided a reasonable estimate of the percentage of human DNA in samples containing known mixtures of human and bacterial DNA. Quantification of buccal DNA from samples collected in a breast cancer case-control study by PicoGreen and real-time PCR indicated that cytobrush and mouthwash DNA samples contain similar percentages of human amplifiable DNA. Real-time PCR is recommended for the quantification of buccal cell DNA in epidemiological studies since it provides precise estimates of human amplifiable DNA across the wide range of DNA concentrations commonly observed in buccal cell DNA samples.
García-Closas
M
M
Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD 20892, USA. garciacm@exchange.nih.gov
Moore
L E
LE
Rabkin
C S
CS
Franklin
T
T
Struewing
J
J
Ginzinger
D
D
Alguacil
J
J
Rothman
N
N
D002610:Cheek; D004247:DNA / Q000032:analysis*; D016021:Epidemiologic Studies; D006801:Humans; D009061:Mouth Mucosa / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet
D004247:DNA