Detection of DNA fragmentation and apoptotic proteins, and quantification of uncoupling protein expression by real-time RT-PCR in adipose tissue

Better understanding of the mechanisms involved in adipose tissue growth and metabolism is critical for the development of more effective treatments for obesity. However, because of its high lipid and low protein content, adipose tissue can present unique problems in some experimental procedures. We describe three protocols that provide new or improved methods for analysis of DNA, RNA, and protein from different adipose tissues. The first protocol provides a simple and rapid method for separation of fragmented DNA and visualization of apoptotic DNA laddering without the need for radioisotopes. This technique allows for an estimate of the amount of DNA fragmentation, and hence, apoptosis. The second protocol details subcellular fractionation of adipose tissue for the extraction of protein in the mitochondrial and cytosol fractions and the measurement of apoptotic protein (Bcl-2 and Bax) levels in each fraction. The last protocol involves extraction of total RNA from adipose tissue and the measurement of uncoupling protein mRNA using real-time RT-PCR, a method that has not previously been used to measure expression of uncoupling proteins in adipose tissue. (C) 2003 Elsevier B.V. All rights reserved.