Quantitation of mRNA Levels by RT-PCR in Cells Purified by FACS: Application to Peripheral Cannabinoid Receptors in Leukocyte Subsets

Cell-sorting and mRNA amplification techniques with RT-PCR have been combined to study the gene expression of cytokines (1 –3 ), receptors (4 –6 ) and differentiation antigens of purified hematopoietic cells (7 –9 ). Nevertheless, a major drawback of these techniques is the difficulty in obtaining enough purified cells. The extended cell-sorting times necessary for this make it unsuitable for routine analysis when cell populations are poorly represented or target genes are weakly expressed. These studies are thus limited to qualitative or semiquantitative approaches to mRNA from sorted cells. Recently, we studied the distribution patterns of central and peripheral cannabinoid receptors (CB2) in human immune tissues and leukocyte subpopulations using a quantitative reverse-transcriptase-polymerase chain reaction (RT-PCR)-based method (10 ). As a part of this study, we report here on a novel technique for quantitative assay of this receptor, applicable to a number of sorted cells as low as 2 � 10⁵ . This method combines quantitative RT-PCR and measurement of PCR products using an enzyme detection system in a 96-well microplate.