HUNT Extraction and concentration normalisation of DNA
HUNT biobank are experienced in procedures involving the extraction of DNA from blood (coagels, whole blood and buffy coat). Primarily by the Gentra Puregene Blood Kit (Qiagen), DNA has been extracted from all samples collected at HUNT2 (samples from participants with diabetes). For blood samples (buffy coat) collected at HUNT3, DNA-extractions have mainly been done using the MasterPure DNA purification Kit for blood, Buffy Coat DNA Purification Protocol (Epicentre). Both extraction methods are based on desalting and are validated both by the supplier and HUNT bioobank.
The recently acquired robotic system for DNA-extraction (Hamilton – chemagic STAR) is presently under optimisation at HUNT biobank. The robot uses technology based on the DNA molecules binding to magnetic beads (M-PVA Magnetic Beads). Future HUNT3 DNA-extractions in addition to extractions from various other sources will use this system.
DNA absorbs light at 260 nm and proteins at 280 nm. At present, Quantity (concentration, derived from A260) and quality / purity (ratio A260/A280) are controlled routinely at the biobank by measuring every eight sample spectrophotometrically by use of NanoDrop (ND1000 or ND8000).
DNA yield - quantity
DNA-yield (µg DNA/ml blood) may be estimated based on the DNA-concentration measured, volumes of TE buffer added and knowledge of the original volume of blood in a sample. DNA-yield varies dependent on quality and number of white blood cells in the blood sample. Theoretical yields are expected in the range of 8-50 µg DNA per ml whole blood. By experience, average DNA yields at the HUNT biobank are ~ 20 µg DNA/ml blood (±10). Fresh samples are expected to give higher yields and our experience is mostly based on long-term stored samples (- 80 °C).
DNA – quality / purity
For samples of good quality A260/A280 is expected to lie in the range of 1.6 – 2.1. Lower values may indicate protein contamination while higher values may indicate a high content of RNA. A A260/A280 ratio of ~ 1.8 defines "pure" samples. At HUNT biobank thae average ratio A260/A280 in general is found to be 1.8 (±0.2).
Concentration normalisation of DNAAt normalisation, dilutions of DNA are performed to a given concentration e.g. 100, 50 or 25 ng/µl. At HUNT biobank, Biomek pipetting robots linked to a fluorometer are employed. Before normalising to a given concentration, all samples concentration measurements are performed using PicoGreen reagents. Every eight normalised sample is spectrophotometrically tested routinely a second time using NanoDrop (ND1000 or ND8000).
HUNT biobank has since 2004 supplied normalised DNA to many projects and for many types of analysis.
800 DNA-samples were normalised to a concentration of 50 ng/µl (PicoGreen technology).
100 of these samples were control tested spectrophotometrically (NanoDrop).
The average spectrophotometric measurements were 50 ng/µl (95 % KI; 38-63 ng/µl).