Abstract
Investigation of the in vivo regulation of gene expression in human subcutaneous white adipose tissue (WAT) relies on the ability to estimate the changes in specific mRNA levels in biopsies taken before and after a designed intervention (i.e., diet, exercise, hyperinsulinemic clamp, and so on). Such study is limited by the size of the samples that could be taken, and by the high lipid content of adipose tissue (AT), which renders the isolation of total RNA difficult. However, it is possible to obtain highly pure RNA preparations from low amounts of AT, using commercially available kits that are based on the selective binding of RNA molecules on silica-gel supports. Nevertheless, the low yield in total RNA with fat tissue requires a highly sensitive method to quantify specific mRNA molecules, in order to estimate the expression levels of the genes of interest. An adequate and powerful method is the quantification of mRNAs by reverse transcription followed by competitive polymerase chain reaction (RT-cPCR), which relies on the addition of a known amount of an exogenous DNA molecule (called competitor) to the amplification mixture, after the reverse transcription step (1–3). In this method, however, the efficiency of the reverse transcription reaction is not controlled. We have therefore determined experimental conditions that allow 100% efficiency of cDNA synthesis during the reverse transcription step (4). This is possible when using a specific antisense primer (see Note 1) and a thermostable reverse transcriptase to perform the reaction at elevated temperature (see Note 2).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ferré, F. (1992) Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods Appl. 2, 1–9.
Gilliland, G., Perrin, S., Blanchard, K., and Bunn, H. F. (1990) Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc. Natl. Acad. Sci. USA 87, 2725–2729.
Bouaboula, M., Legoux, P., Pességué, B., Delpech, B., Dumont, X., Piechaczyk, M., Casellas, P., and Shire, D. (1992) Standardization of mRNA titration using a polymerase chain reaction method involving co-amplification with a multispecific internal control. J. Biol. Chem. 267, 21,830–21,838.
Auboeuf, D. and Vidal, H. (1997) Use of the reverse transcription-competitive polymerase chain reaction to investigate the in vivo regulation of gene expression in small tissue samples. Anal. Biochem. 245, 141–148.
Raeymaekers, L. (1995) Commentary on the practical applications of competitive PCR. Genome Res. 5, 91–94.
Lefebvre, A. M., Laville, M., Vega, N., Riou, J. P., van Gaal, L., Auwerx, J., and Vidal, H. (1998) Depot-specific differences in adipose tissue gene expression in lean and obese subjects. Diabetes 47, 98–103.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2001 Humana Press Inc.
About this protocol
Cite this protocol
Vidal, H. (2001). Quantification of Lipid-Related mRNAs by Reverse Transcription-Competitive Polymerase Chain Reaction in Human White Adipose Tissue Biopsies. In: Ailhaud, G. (eds) Adipose Tissue Protocols. Methods in Molecular Biology™, vol 155. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-231-7:083
Download citation
DOI: https://doi.org/10.1385/1-59259-231-7:083
Publisher Name: Springer, Totowa, NJ
Print ISBN: 978-0-89603-747-2
Online ISBN: 978-1-59259-231-9
eBook Packages: Springer Protocols