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MB Sample ID: SA033569

Local Sample ID:OHSUserum_PO6716_3
Subject ID:SU000631
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Species Group:Human

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Sample Preparation:

Sampleprep ID:SP000638
Sampleprep Summary:For the serum samples, 10 mL of whole blood was drawn and centrifuged so the serum could be extracted. For the DBS samples, 500 μL of blood was collected and 125 μL was blotted in four areas on a Whatman FTA Classic Card. Polar metabolites and lipid extracts were derived from the same sample type (DBS or serum) using a modified Folch extraction in June 2015 and analyzed with MS shortly after. The DBS punch from each patient was transferred into to a 2.0 mL tube where 50 μL of water and then 1200 μL of –20°C 2:1 chloroform/methanol were added. Each sample was vortexed for 30 s then transferred into a shaker at 22°C for 60 min at 600 rpm. The samples were vortexed again for 30 s and then 250 μL of water was added to induce a phase separation. The sample was gently inverted several times, placed at room temperature for 5 min and then centrifuged at 10,000 g for 5 min at 4°C and put on ice to maintain the clear phase separation. Finally, 400 μL of the top polar layer was removed, dried in a Speedvac, and stored at –80°C for analysis of polar metabolites, while 700 μL of the bottom nonpolar layer was removed, dried in a Speedvac, and stored at –20°C in 250 μL of 2:1 chloroform/methanol for lipid analyses. Serum lipids were extracted using a similar procedure except 25 μL of serum was used and then 600 μL of –20°C 2:1 chloroform/methanol was added. After vortexing and shaking, 125 μL of water was added to induce a phase separation and 200 μL of the top polar layer and 350 μL of the bottom nonpolar lipid layer were removed and stored as outlined above. Prior to analysis, the total lipid extracts were dried down and then reconstituted in 70 μL and 100 μL of MeOH for the DBS and serum samples, respectively. To generate pooled case and control samples for LC/MS/MS and LC/IMS-MS analyses, 5 μL aliquots from each reconstituted DBS and serum sample were removed and combined.