Core B Molecular Biology and Lipidomics provides multiple essential services to every investigator working on this Program Project Grant. These services include routine experimental procedures as well as unique assays that provide unparalleled and comprehensive molecular insight into lipid metabolism. The core is directed by Dr. David W. Russell, who has served as its Principal Investigator for the past 30 years. Routine services include protein gel electrophoresis and immunoblotting; DNA sequencing of wild-type and mutant cDNAs and plasmid constructs; RNAseq assays to assess the transcriptomes of cells and tissues; quantitative PCR assays to measure mRNA levels expressed from hundreds of mouse, human, and hamster genes; the design and ordering of custom oligonucleotide probes and primers; the preparation of genomic and plasmid DNAs for experimentation and storage; and the assessment of physiological parameters (oxygen consumption, carbon dioxide production, hemodynamic parameters, weight gain and loss, activity, etc.) in living animals. Unique assays developed through the auspices of this Program Project include mass spectrometry- based quantification of individual lipid species including sterols, oxysterols, ceramides, steroids, bile acids, and fatty acids; lipidomic profiling that yields qualitative to semi-quantitative assessments of thousands of lipid molecular species; and flux measurements that quantify the flow of metabolites through a designated biosynthetic or catabolic pathway in cultured cells and living animals. The extensive and expensive equipment needed to provide these services, including multiple mass spectrometers, liquid-handling robots, metabolic cages, freezers, real-time PCR machines, and routine and next-generation DNA sequencers, is provided solely by the institution and represents a substantial cost-sharing investment. The services provided by this Core play a major role in every specific aim in this Program Project and in every publication that arises from the completed research. C/PPG 2015 – Core B – Project Summary