In December 2007, collection of a saliva sample using the Genotek Oragene-DNA Self-Collection Kit was initiated. Oragene-DNA yields high-quality, high-quantity (@ 110ug) DNA from a small saliva sample. Oragene-DNA is optimized to preserve and stabilize saliva samples for long term storage at room temperature without DNA degradation. DNA from Oragene-DNA is equivalent to DNA from blood and has been successfully used for PCR and genotyping in genome-wide Association studies.
Participants are sent a letter, informed consent, collection instructions and saliva collection kit in the mail. The participant is asked to return the sample in the collection kit to the Coordinating Center. Upon receipt of the sample in the Coordinating Center, the collection kit is weighed and the container is assessed for cracks, leaks, or other damage. Signed informed consents are stored in a locked file cabinet. An accession number is assigned to the collection kit. Samples are sent to Cincinnati for processing and storage in the Molecular Genetics Laboratory.
Methods of DNA extraction and storage
On receipt in the laboratory the sample is immediately labeled with a sample accession number which is recorded with the subject's study ID number in a computer log and in a sample accession book. Accession numbers are used to track samples; at no time after arrival in the laboratory are subject names attached to the sample. Samples are processed according to Oragene-DNA procedures supplied by the company, DNA Genotek. When samples are ready to be processed, they are incubated at 50 degrees C in a water incubator for a minimum of 1 hour or in an air incubator for a minimum of 2 hours. 500ul of the mixed Oragene-DNA/saliva sample is transferred to a 1.5ml microcentrifuge tube. 20ul of Oragene-DNA Purifier is added to the microcentrifuge tube and mixed by vortexing for a few seconds. The sample is incubated on ice for 10 minutes, then centrifuged at room temperature for 5 minutes at 13,000 rpm (15,000 x g). The clear supernatant is carefully transferred with a pipet into a fresh microcentrifuge tube. 500ul of room-temperature 95-100% ethanol is added to 500ul of supernatant and gently mixed by inversion 10 times. The sample is allowed to stand for 10 minutes at room air to allow the DNA to fully precipitate. The tube is then placed in the centrifuge for 2 minutes at room air at 13,000 rpm (15,000 x g). Supernatant is then removed with a pipet and discarded taking care to avoid disturbing the DNA pellet. 100ul of DNA butter is added to dissolve the DNA pellet and vortex for at least 5 seconds. Additional steps may be used to ensure complete hydration of the DNA.