Return to study ST001106 main page

MB Sample ID: SA075243

Local Sample ID:1610-16
Subject ID:SU001151
Subject Type:Mammal
Subject Species:Ovis aries
Taxonomy ID:9940
Age Or Age Range:within 6 hours of birth

Select appropriate tab below to view additional metadata details:

Sample Preparation:

Sampleprep ID:SP001158
Sampleprep Summary:Homogenized right ventricle tissue samples (100 mg from 7 fetuses and 7 newborns) were used for lipid extraction via the Folch method. Briefly, 2 mL of internal standard mixture was added to each sample before adding 1.2 mL of 2:1 chloroform/methanol solution (HPLC-grade). Samples were incubated at 4 °C for 20 min with occasional vortexing. Following incubation, 200 mL of water (HPLC-grade) was added and samples were incubated at 4 °C for 10 min. Samples were centrifuged at 2,000 g for 5 min at 4 °C, and the resulting chloroform layer was removed and transferred into a clean tube. The extraction process was repeated with 400 mL 2:1 chloroform/methanol (HPLC-grade) and the resulting chloroform layers were combined. Samples were dried under a nitrogen stream at 30 °C and stored at -80 °C until reconstitution. Samples were reconstituted with 200 mL of isopropanol and 2 mL injection standard mixture. Ammonium acetate and all analytical grade solvents (formic acid, chloroform, and methanol) were purchased from Fisher Scientific (Waltham, MA, USA). All mobile phase solvents were Fisher Optima LC/MS-grade (acetonitrile, isopropanol, and water). Organ: Heart, Organ Specification: Right Ventricle
Processing Method:50 mg of tissue was weighed and added to tubes with three 3mm glass beads, two 5mm steal beads, and 0.7mm zirconia beads (“2 squirts”). HPLC-water (1mL) was added to each tube before homogenization at 1800 rpm for 30 seconds for 5 rounds. Samples were incubated at 4C for 20 min between rounds. Following homogenization, samples were centrifuged at 2,000g for 10 min at 4C to pellet tissue debris before transferring supernatant to a clean tube. Homogenates were stored at -80C.
Extraction Method:2 mL of internal standard mixture was added to each sample before adding 1.2 mL of 2:1 chloroform/methanol solution (HPLC-grade). Samples were incubated at 4 °C for 20 min with occasional vortexing. Following incubation, 200 mL of water (HPLC-grade) was added and samples were incubated at 4 °C for 10 min. Samples were centrifuged at 2,000 g for 5 min at 4 °C, and the resulting chloroform layer was removed and transferred into a clean tube. The extraction process was repeated with 400 mL 2:1 chloroform/methanol (HPLC-grade) and the resulting chloroform layers were combined. Samples were dried under a nitrogen stream at 30 °C (Organomation Associated MultiVap) and stored at -80 °C until reconstitution.
Sample Resuspension:Samples were reconstituted with 200 mL of isopropanol and 2 mL injection standard mixture and vortexed to mix. Fifty uL of reconstituted sample was transferred to a glass LC vial for analysis.
Sample Spiking:Internal standards: lysophosphatidylcholine (17:0), phosphatidylcholine (17:0/17:0), phosphatidylglycerol (14:0/14:0), phosphatidylethanolamine (15:0/15:0), phosphatidylserine (14:0/14:0), triglyceride (15:0/15:0/15:0). Injection Standards: lysophosphatidylcholine (19:0), phosphatidylcholine (19:0/19:0), phosphatidylglycerol (17:0/17:0), phosphatidylethanolamine (17:0/17:0), phosphatidylserine (17:0/17:0), triglyceride (17:0/17:0/17:0).
  logo