title: Isolation of Mature Adipocytes from White Adipose Tissue and Gene Expression Studies by Real-Time Quantitative RT-PCR.
abstract: The study of adipose tissue and more specifically of adipocytes is considered pivotal for dissecting molecular mechanisms responsible for alterations in several organs and systems, including adipose tissue, not only in obesity but also in other diseases (hypertension, heart failure). Adipose tissue is a complex tissue composed of adipocytes and the stromal vascular fraction which includes a heterogeneous population of preadipocytes, blood cells, endothelial cells, and macrophages. In the present chapter, methods are detailed to generate purified mature adipocytes from white adipose tissue by using enzymatic digestion. Such methods should help laboratories to study the specific roles of adipocytes in different pathologies and are easily adaptable to different animal models. Moreover, as gene activity is controlled at both transcriptional and posttranscriptional levels, it is very important to determine the levels of messenger ribonucleic acid (mRNA) of genes of interest. This process involves the isolation of total RNA and subsequent analysis of the mRNA of interest by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Given the unique nature of adipose tissue and adipocytes (i.e., containing high amounts of lipid), we have set up a special RNA isolation technique in both white adipose tissue and isolated mature adipocytes from mice. In summary, isolation and culture of adipocytes in vivo and gene expression studies will help to understand the mechanisms that control adipocyte function in physiological and pathological states and may lead to design interventions that might affect the adipocyte birth-death balance or phenotype.
authors.affiliation: Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, 126 University Place, Glasgow, G12 8TA, Scotland, UK. cattuong.ndc@gmail.com.
authors.forename: Aurelie Nguyen Dinh
authors.initials: AN
authors.lastname: Cat
authors.affiliation: Department of Pharmacology, School of Medicine, Instituto de Investigación Hospital Universitario La Paz (IdiPAZ), Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029, Madrid, Spain. ana.briones@uam.es.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Briones
mesh_terms: D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism*; D000273:Adipose Tissue / Q000166:cytology*; D052436:Adipose Tissue, White / Q000166:cytology* / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
keywords: Mature adipocytes isolation; Total RNA; White adipose tissue; qRT-PCR