Johns Hopkins All Children's CRIB / MDC

Sample Preparation Documents

Overview

Depending on the type of biological sample – tissue, serum, or cells, – different preliminary sample preparation is needed, as described briefly below.  Certain sample types require more preparation, and in some cases specialized equipment.  As a result, there are certain samples the MDC may not equipped to support.  If an individual user lab has the resources to appropriately prepare samples, MDC staff are happy to discuss these on a case-by-case basis.

Liquid Biological Samples

Biological fluids (plasma, serum, CSF, and urine) are prepared directly via a liquid-liquid extraction that is described below, or using solid phase extraction. 

Tissue

Frozen samples are weighed, then pulverized to a fine powder (freeze-fracturing). Following pulverization the powdered tissue is transferred to a chilled Eppendorf tube, and ice-cold methanol is added to quench metabolism. Sample is then vortexed, pulse-sonicated, and extracted using liquid-liquid or solid phase extraction.

Cultured Cell Samples

Cells must be separated from media prior to being flash frozen and shipped to the MDC. Upon reception cells are thawed, pulse-sonicated, and subjected to liquid-liquid or solid phase extraction.

Liquid-Liquid Extraction

Our liquid-liquid extraction involves three sequential extractions at different pH values.  First, a neutral pH extraction is performed, and an aliqout of organic phase is saved for lipids.  The organic phase is re-extracted under acidic condition and then again under basic conditions. This allows for the most effective removal of all classes of metabolites.  The resulting final organic layer contains hydrolyzed lipids, but is still useful for different lipid class analysis.  Intact lipids are analyzed from the neutral pH extraction.  Prior to analysis, all aqueous phases are then dried down and resuspended together in the LC-MS mobile phase for analysis.  Different aliquots are generated for each chromatography method used.