DNA extraction from human tissues

2.7.1 Homogenisation of frozen skeletal muscle

Before beginning the tissue homogenisation, an empty 1.5ml microcentrifuge tube was weighed. Muscle was placed in a porcelain mortar, chilled with liquid nitrogen, and manually ground with a porcelain pestle. Liquid nitrogen was used to keep the samples and

instruments cool throughout the process. Powdered tissue was transferred to a 1.5ml microcentrifuge tube using a chilled scalpel. The microcentrifuge tube was then re-weighed and the sample mass calculated. A maximum of 25mg of muscle tissue was accepted for each 1.5ml microcentrifuge tube, with any excess removed to another tube. In between stages of this process, muscle tissue samples were kept in liquid nitrogen.

2.7.2 DNA purification from frozen skeletal muscle

Isolation of DNA from homogenised skeletal muscle samples was performed using a QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s guidelines as outlined in the ‘DNA Purification from Tissues’ protocol in the QIAamp DNA Mini and Blood Mini Handbook (06/2012). For the final elution stage, 100μl Buffer AE were added to the centre of the membrane and allowed to incubate at room temperature for five minutes before centrifugation at full speed for one minute.

Temperature Duration

Activation of proteinase K enzyme 55°C 3 hours

Deactivation of proteinase K enzyme 95°C 10 minutes

62 2.7.3 DNA purification from urine

Urine samples were collected in 50ml Falcon tubes and centrifuged at 160rcf for 10 minutes to generate a pellet of urinary sediment. The supernatant was removed and the pellet was re-suspended in 1ml sterile PBS. DNA was extracted from the urinary sediment using a QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s guidelines, outlined in the protocol ‘Isolation of Genomic DNA from Urine’ (QIAamp DNA Micro Handbook 12/2014). For elution of DNA from the spin column, 50μl of Buffer AE were added to the centre of the

membrane and left to incubate at room temperature for five minutes before centrifugation at full speed for one minute.

2.7.4 DNA purification from buccal swabs

Dacron buccal swabs were separated from the stick using scissors into a 2ml microcentrifuge tube. DNA was extracted from buccal swabs using a QIAamp DNA Mini Kit (Qiagen) as

outlined in the manufacturer’s guidelines ‘DNA purification from Buccal Swabs (Spin Protocol)’ (QIAamp DNA Mini and Blood Mini Handbook 05/2016). Following the spin column purification, outlined in steps 1-9 of the protocol, 100μl buffer AE were added for elution of DNA instead of 150μl. This was to increase the final DNA concentration.

2.7.5 DNA purification from hair

Hair samples were cut to approximately 2cm lengths into 2ml microcentrifuge tubes with 150μl sterile PBS. DNA was isolated from these samples using the QIAamp DNA Micro Kit (Qiagen) by adapting the manufacturer’s protocol for ‘Isolation of Genomic DNA from Laser- Microdissected Tissues’ (QIAamp DNA Micro Handbook 12/2014). 300μl buffer ATL were added along with 20μl proteinase K and the sample was mixed by pulse-vortexing for 15 seconds before being placed in a thermomixer to incubate at 56°C for three hours. Following centrifugation to remove drops from the lid, a further 250μl buffer ATL were added to the sample with 50μl buffer AL. The solution was thoroughly mixed by pulse vortexing for 15 seconds to ensure sufficient lysis. 50μl 10% ethanol were added to the sample, which was then mixed by pulse-vortexing and incubated for five minutes at room temperature. Again, the sample was centrifuged to remove any liquid from the cap. The lysate was transferred to the QIAamp MinElute column without wetting the rim and centrifuged at 5510rcf for one minute. Flow-through was discarded and the spin column placed in a clean 2ml collection tube. 500μl Buffer AW1 were added to the spin column and then centrifuged at 5510rcf for

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one minute. After discarding flow through and placing the column in a clean collection tube, the step was repeated with 500μl Buffer AW2. To dry the membrane, the spin column was centrifuged at full speed (16873rcf) for three minutes while empty. The column was then transferred to a clean 1.5ml microcentrifuge tube and 50μl Buffer AE added to the centre of the membrane. This was left to incubate at room temperature for five minutes to maximise the DNA yield before a final centrifugation step at 16873rcf for one minute.

2.7.6 DNA purification from whole blood

Isolation of DNA from human blood samples was performed using a QIAamp DNA Mini Kit (Qiagen). 40μl proteinase K were added to 600μl whole blood sample along with 600μl Buffer AL. After pulse vortexing for 15 seconds to mix, the sample was incubated at 56°C for 10 minutes while shaking using a Thermomixer (Eppendorf). Droplets were removed from the lid by centrifugation and 600μl 100% ethanol added to the sample before pulse-

vortexing and centrifugation once more. 600μl sample was transferred to the spin column, without wetting the rim, and centrifuged at 5510rcf for one minute. This step was repeated until the entire sample had been passed through the spin column. The remaining steps follow the manufacturer’s protocol as outlined in the QIAamp DNA Mini and Blood Mini Handbook (05/2016), eluting into a final volume of 100µl Buffer AE.

2.7.7 DNA concentration and storage

The concentration of DNA extracted from homogenate tissues was measured using the Nanodrop ND-1000 spectrophotometer (Labtech International). Extracted DNA was stored as 10µl aliquots at -20°C, or -80°C for long-term storage.