Quantitative method to determine mRNA levels by reverse transcriptase-polymerase chain reaction from leukocyte subsets purified by fluorescence-activated cell sorting: Application to peripheral cannabinoid receptors
✍ Scribed by J. Marchand; A. Bord; G. Pénarier; F. Lauré; P. Carayon; P. Casellas
- Book ID
- 101243036
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 145 KB
- Volume
- 35
- Category
- Article
- ISSN
- 0196-4763
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✦ Synopsis
Background: While cytometry is widely used in the detection of cell proteins, its application to quantitative evaluation remains problematic when target proteins or receptors are weakly expressed in cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) is a technique whose sensitivity and specificity make it appropriate for analyzing nucleic acids and thus genes expressed in cells. Combining these two techniques, we developed a method to quantify the transcript expression of the peripheral cannabinoid receptor (CB2-r) in peripheral blood lymphocyte subpopulations and in tonsillar B-cell subpopulations. Methods: This strategy first involves quantitative RT-PCR performed kinetically, followed by enzyme detection of PCR products using an oligonucleotide probe sandwichhybridization assay onto microplates.
Results: B cells exhibit CB2-receptor mRNA levels 10 times higher than those of other lymphocyte subsets. Using this technique, we observed a modulation of CB2-r mRNA level following tonsillar B-cell differentiation. Lastly, this new technology was validated by comparing the mRNA levels of CB2-r with the expression of CB2-r proteins assayed by flow cytometry, using specific CB2-r antibody labelling. Conclusions: This method allows precise measurement of the mRNA of CB2-r performed on cell numbers as low as 10 5 after sorting. Its performance, high accuracy, reproducibility, and reliability make it a valuable tool for assaying proteins weakly expressed in cells. Cytometry 35:227-234, 1999. 1999 Wiley-Liss, Inc.