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) and saliva.
HUNT2 DNA has primarily been extracted using the Gentra Puregene Blood Kit (Qiagen).
For HUNT3 samples, DNA-extractions have been done using Buffy Coat DNA Purification Protocol (Epicentre) by MasterPure DNA purification Kit for blood. Both extraction methods are based on desalting.
The recently acquired robotic system for DNA-extraction (Hamilton – Chemagic STAR) is primarily used for HUNT3 samples. This system is based on Chemagen`s Magnetic Bead technology (PerkinElmer).
HUNT Biobank can also process external samples using validated methods for extraction.
DNA yield varies dependent on quality and number of cells in the sample. For whole blood, 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 yield are controlled routinely by measuring every eight sample spectrophotometrically.
Normalisation of DNA concentration
At normalisation, DNA is diluted to give concentrations of e.g. 50 or 25 ng/µl dependent on the original sample concentration. At HUNT Biobank, Biomek pipetting robots are used for normalisation. Before normalising to a given concentration, all concentration measurements are performed fluorometric using PicoGreen reagents.
After normalisation of samples to 50 ng/µl, a mean concentration of 50.19 (± 9.9) ng/µl has been shown (based on the measurement of 1255 samples).
DNA – quality / purity
Quality / purity (ratio A260/A280) are controlled routinely by measuring every eight sample spectrophotometrically.
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. At HUNT Biobank the average ratio A260/A280 in general is found to be 1.8 (±0.2).