Maximizing DNA yield for epidemiologic studies: no more buffy coats?

Some molecular analyses require microgram quantities of DNA, yet many epidemiologic studies preserve only the buffy coat. In Frederick, Maryland, in 2010, we estimated DNA yields from 5 mL of whole blood and from equivalent amounts of all-cell-pellet (ACP) fraction, buffy coat, and residual blood cells from fresh blood (n = 10 volunteers) and from both fresh and frozen blood (n = 10). We extracted DNA with the QIAamp DNA Blood Midi Kit (Qiagen Sciences, Germantown, Maryland) for silica spin column capture and measured double-stranded DNA. Yields from frozen blood fractions were not statistically significantly different from those obtained from fresh fractions. ACP fractions yielded 80.6% (95% confidence interval: 66, 97) of the yield of frozen whole blood and 99.3% (95% confidence interval: 86, 100) of the yield of fresh blood. Frozen buffy coat and residual blood cells each yielded only half as much DNA as frozen ACP, and the yields were more variable. Assuming that DNA yield and quality from frozen ACP are stable, we recommend freezing plasma and ACP. Not only does ACP yield twice as much DNA as buffy coat but it is easier to process, and its yield is less variable from person to person. Long-term stability studies are needed. If one wishes to separate buffy coat before freezing, one should also save the residual blood cell fraction, which contains just as much DNA.

Long-term storage and recovery of buccal cell DNA from treated cards.

Economical methods for collecting and storing high-quality DNA are needed for large population-based molecular epidemiology studies. Buccal cell DNA collected via saliva and stored on treated filter paper cards could be an attractive method, but modest DNA yields and the potential for reduced recovery of DNA over time were unresolved impediments. Consequently, buccal cell DNA collection via oral mouthwash rinsing became the method of choice in epidemiologic studies. However, the amount of genomic DNA (gDNA) required for genotyping continues to decrease, and reliable whole genome amplification (WGA) methods further reduced the mass of gDNA needed for WGA to 10 ng, diminishing the obstacle of low DNA yields from cards. However, concerns about yield and DNA quality over time remained. We located and analyzed 42 buccal cell saliva samples collected and stored on treated cards for 7 years at room temperature, -20 degrees C, and -80 degrees C. We recovered DNA from the treated cards, estimated the concentration by a human-specific quantitative real-time PCR assay, and evaluated the quality by PCR amplification of 268-, 536-, and 989-bp fragments of the beta-globin gene and by AmpFlSTR Identifiler assay analysis. Most DNA yields per 3-mm punch were <10 ng, and most PCR amplicons failed to amplify, where size of the amplicon was negatively associated with successful amplification. Using these methods, treated cards did not consistently provide sufficient quantities of buccal cell gDNA after 7 years of storage for genotyping or WGA.

DNA banking for epidemiologic studies: a review of current practices.

To study genetic risk factors for common diseases, researchers have begun collecting DNA specimens in large epidemiologic studies and surveys. However, little information is available to guide researchers in selecting the most appropriate specimens. In an effort to gather the best information for the selection of specimens for these studies, we convened a meeting of scientists engaged in DNA banking for large epidemiologic studies. In this discussion, we review the information presented at that meeting in the context of recent published information. Factors to be considered in choosing the appropriate specimens for epidemiologic studies include quality and quantity of DNA, convenience of collection and storage, cost, and ability to accommodate future needs for genotyping. We focus on four types of specimens that are stored in these banks: (1) whole blood preserved as dried blood spots; (2) whole blood from which genomic DNA is isolated, (3) immortalized lymphocytes from whole blood or separated lymphocytes, prepared immediately or subsequent to cryopreservation; and (4) buccal epithelial cells. Each of the specimens discussed is useful for epidemiologic studies according to specific needs, which we enumerate in our conclusions.