RNA isolation and real-time quantitative RT-PCR.

Adipose tissue has emerged as a major endocrine organ producing a wide spectrum of hormones and factors that play crucial roles in regulating cell turnover and function, not only locally within the adipose tissue but also in the brain and other key metabolic organ systems. It is known that gene activity is controlled at both transcriptional and post-transcriptional levels. Consequently, one of the most important means by which the activity of a gene is assessed is through the determination of levels of the corresponding messenger ribonucleic acid (mRNA). This process involves the isolation of total cellular RNA and subsequent analysis of the mRNA of interest. Given the unique nature of adipose tissue and adipocytes (i.e., containing high amounts of lipid), special RNA isolation techniques that have been tested in both white adipose tissue and isolated mature adipocytes from rats and mice will be presented. Although several methods are available for mRNA quantitation, we will describe a real-time quantitative reverse transcription polymerase chain reaction protocol because of its superior sensitivity and reliability.