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Comparative Study
, 35 (3), 227-34

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

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Comparative Study

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

J Marchand et al. Cytometry.

Abstract

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 sandwich-hybridization 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.

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