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

Background: While cytometry is widely used in the detection of cell protein s, its application to quantitative evaluation remains problematic when targ et proteins or receptors are weakly expressed in cells. Reverse transcripta se-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 t o quantify the transcript expression of the peripheral cannabinoid receptor (CB2-r) in peripheral blood lymphocyte subpopulations and in tonsillar B-c ell subpopulations. Methods: This strategy first involves quantitative RT-PCR performed kinetic ally, 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 thos e of other lymphocyte subsets. Using this technique, we observed a modulati on of CB2-r mRNA level following tonsillar B-cell differentiation. Lastly, this new technology was validated by comparing the mRNA levels of CB2-r wit h the expression of CB2-r proteins assayed by flow cytometry, using specifi c CB2-r antibody labelling. Conclusions: This method allows precise measurement of the mRNA of CB2-r pe rformed on cell numbers as low as 10(5) after sorting. Its performance, hig h accuracy, reproducibility, and reliability make it a valuable tool for as saying proteins weakly expressed in cells. (C) 1999 Wiley-Liss,Inc.