Quantification of Lipid-Related mRNAs by Reverse Transcription-Competitive Polymerase Chain Reaction in Human White Adipose
Tissue Biopsies
By: Hubert Vidal2
| Abstract |
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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).
Book Title: Adipose Tissue Protocols
Series: Methods in Molecular Biology | Volume: 155 | Pub. Date: Mar-01-2001 | Page Range: 83-88 | DOI: 10.1385/1-59259-231-7:083
Subject: Cell Biology
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