ABSTRACT
The expression rate of key genes in specific cell populations in vivo, such as obtained from surgical biopsies, is of great interest. The polymerase chain reaction (PCR) technique1 in combination with a cDNA synthesis step using reverse transcriptase2 can be used to detect very low numbers of mRNA copies. Several methods also allow reliable quantitation of small amounts of mRNA.3 In order to measure rate of expression in cellular samples, the quantitative estimates should most appropriately be related to the number of cells in the original sample. Cell counting in small samples, however, constitutes a methodological problem. Modern flow cytometers are commonly used to measure cell surface proteins after antibody staining and can also sort cells according to specific cell surface markers. These features led us to test whether flow cytometry could be used in conjunction with quantitative RT-PCR for the estimation of mRNA copies per cell. Freshly isolated endothelial cells were sorted by flow cytometry and then lysed. Endothelial cells in primary culture were lysed directly in the microtiter plate wells. The number of cells in the lysate was determined by counting of nuclei after propidium iodide staining using flow cytometry. Plasminogen activator inhibitor-1 (PAI-1), an important regulator of the fibrinolytic system,5 is expressed by endothelial cells and was used here as an example target molecule. The number of PAI-1 mRNA copies per cell was determined by quantitative RT-PCR using point-mutated PAI-1 cRNA as an internal standard, although other quantitative RTPCR methods could also be combined with the quantitative flow cytometry. The method allowed reliable and reproducible estimation of the number of target mRNA copies per cell from original cell samples containing less than 1000 cells. This method can be used for the quantitative determination of mRNA in specified cell populations from small tissue samples or cultured cells.
