Collection of buccal cell DNA in seventh-grade children using water and a toothbrush.

We developed a simple and effective method for collecting a large quantity of buccal cell DNA in school-based studies of seventh-grade and older children. Seventh-grade students at schools in Wuhan, China brushed each buccal surface with a soft toothbrush and then rinsed with 10 ml of water. We added 5 ml of 99% ethanol to preserve the sample. Among 1563 samples transported at room temperature over 1 week and then stored for 13-14 months at -70 degrees C before extraction, using a modified Gentra Puregene protocol, the median total DNA yield was 108 microg, range of 14 to 416 microg. We assayed every 20th sample (n = 77) for NAT2 by the PCR, and all samples gave a 1093-bp product. From the 1563 samples, we obtained a result for single nucleotide polymorphisms in the interleukin-13 gene (at +2044) by RFLP-PCR on 98.8% and in the promoter of the myeloperoxidase gene (at -463) by real-time PCR on 99.7%. A water-rinse method, that we used among 12th-grade students in Southern California, gave a lower total DNA yield than the toothbrush rinse (median of 17 microg) and a slightly reduced ability to generate a PCR product. However, 26 of 27 water-rinse samples gave a result for two genes, albumin and CYP1A1, using real-time PCR methods. We did not quantify human, versus bacterial, DNA in our samples. However, given the amounts of total DNA required for genotyping, a sample with the median yield of 108 microg should suffice for approximately 2160 genotypes by RFLP-PCR methods or five times as many by real-time PCR. We recommend the toothbrush-rinse method, combined with a modified Gentra Puregene DNA extraction protocol, for large-scale, in-person collections of buccal cell DNA in children. The method requires only inexpensive, readily available materials and produces a large quantity of high-quality DNA for PCR analyses.

Adolescent smoking in Wuhan, China: baseline data from the Wuhan Smoking Prevention Trial.

BACKGROUND: This study reports the prevalence of adolescent smoking in the urban and rural areas of Wuhan, China, the capital of Hubei Province, on the Yangtze River in central China. METHODS: Smoking behavior was examined by age, gender, and urbanicity as part of the Wuhan Smoking Prevention Trial. Subjects included 6994 seventh- to ninth-grade students attending 22 randomly selected schools in urban and rural districts. Outcome measures included lifetime smoking, past-30-day smoking, established smoking (>100 cigarettes in lifetime), and susceptibility to smoking (absence of a firm commitment not to smoke). RESULTS: Lifetime smoking prevalence was 47% among boys and 18% among girls. Past-30-day smoking prevalence was 16% among boys and 4% among girls. Established smoking prevalence was 2% among boys and 0% among girls. The prevalence of susceptibility to smoking was 31% among boys and 10% among girls. Smoking increased significantly with age (p<.0005). Susceptibility was more prevalent in rural areas than in urban areas (p<.05), but there were no urban-rural differences in lifetime, past 30-day smoking, or established smoking. Trend analyses revealed that smoking increased with age more rapidly among boys than among girls (p<.05). Smoking was more prevalent among rural boys than among urban boys, but it was more prevalent among urban girls than among rural girls (p<.05). CONCLUSIONS: Adolescent smoking is a significant public health problem in China. Boys are at particularly high risk, as are girls living in urban areas. Effective smoking prevention programs for adolescents, as well as restrictions on tobacco industry marketing and youth access to tobacco, are needed to prevent tobacco-related morbidity and mortality in China.