Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA207521

Local Sample ID:	31
Subject ID:	SU002248
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Sample Preparation:

Sampleprep ID:	SP002254
Sampleprep Summary:	Extraction of mouse retina lipids was carried out using a biphasic solvent system of cold methanol, methyl tert-butyl ether (MTBE), and PBS using the extraction method by Matyash et al. (J Lipid Res 2008, 49, (5), 1137-46). In a randomized sequence, tissue lipids (~10 mg) were extracted in bead-mill tubes (ceramic 1.4 mm, Mo-Bio, Qiagen, Germantown, MD) containing a solution of 225 mL MeOH, 750 mL MTBE, and internal standards (Lipid standard Mouse SPLASH LipidoMix at 10 mL per sample, Avanti Polar Lipids, Alabaster, AL). Samples were homogenized in one 30 second cycle and rested on ice for 1 hour with occasional vortexing. Then, 188 mL of PBS was added followed by a brief vortex. Samples were then centrifuged at 14,000 x g for 10 minutes at 4 °C, and the upper phases were collected. Another aliquot of 750 mL MTBE was added to the bottom aqueous layer followed by a brief vortex. Samples were then centrifuged at 14,000 x g for 10 minutes at 4 °C, the upper phases were combined and evaporated to dryness under speedvac. Lipid extracts were reconstituted in 250 mL of mobile phase B and transferred to an LC-MS vial for analysis. Concurrently, a process blank sample was prepared and then a pooled quality control (QC) sample was prepared by taking equal volumes (~50 mL) from each sample after final resuspension. Injection volumes of 2 uL for positive and 10 uL for negative mode, and iterative, tandem mass spectrometry was conducted using the same LC gradient at collision energies of 20 V and 27.5 V, respectively.

Sampleprep ID:

SP002254

Sampleprep Summary:

Extraction of mouse retina lipids was carried out using a biphasic solvent system of cold methanol, methyl tert-butyl ether (MTBE), and PBS using the extraction method by Matyash et al. (J Lipid Res 2008, 49, (5), 1137-46). In a randomized sequence, tissue lipids (~10 mg) were extracted in bead-mill tubes (ceramic 1.4 mm, Mo-Bio, Qiagen, Germantown, MD) containing a solution of 225 mL MeOH, 750 mL MTBE, and internal standards (Lipid standard Mouse SPLASH LipidoMix at 10 mL per sample, Avanti Polar Lipids, Alabaster, AL). Samples were homogenized in one 30 second cycle and rested on ice for 1 hour with occasional vortexing. Then, 188 mL of PBS was added followed by a brief vortex. Samples were then centrifuged at 14,000 x g for 10 minutes at 4 °C, and the upper phases were collected. Another aliquot of 750 mL MTBE was added to the bottom aqueous layer followed by a brief vortex. Samples were then centrifuged at 14,000 x g for 10 minutes at 4 °C, the upper phases were combined and evaporated to dryness under speedvac. Lipid extracts were reconstituted in 250 mL of mobile phase B and transferred to an LC-MS vial for analysis. Concurrently, a process blank sample was prepared and then a pooled quality control (QC) sample was prepared by taking equal volumes (~50 mL) from each sample after final resuspension. Injection volumes of 2 uL for positive and 10 uL for negative mode, and iterative, tandem mass spectrometry was conducted using the same LC gradient at collision energies of 20 V and 27.5 V, respectively.