Quantitative analysis of specific mRNA species in minute cell samples by RT-PCR and flow cytometry.
A method for the quantitative determination of specific mRNAs in small numbers of cells, freshly isolated from tissues or early cell cultures, was developed by combining quantitative reverse transcription-polymerase chain reaction (RT-PCR) and quantitative flow cytometry. Freshly isolated umbilical vein endothelial cells were sorted by flow cytometry and then lysed. The number of cells in the lysate was determined by counting of nuclei after propidium iodide staining using flow cytometry. The number of plasminogen activator inhibitor-1 (PAI-1) mRNA copies per cell was determined by quantitative RT-PCR using point-mutated PAI-1 cRNA as an internal standard. The cells were shown to contain 400-900 copies of PAI-1 mRNA molecules per cell which confirms that endothelial cells in vivo express PAI-1. PAI-1 mRNA expression was also analyzed in small numbers of endothelial cells in primary culture in basal conditions and after incubation with different interleukins. The method allowed reliable and reproducible estimation of the number of mRNA copies per cell from original cell samples containing less than 1000 cells. This method can be used for the quantitative determination of various mRNA species in specified cell populations from small tissue samples or cultured cells.
Cardiovascular Research Unit, Centre for Molecular Medicine, Karolinska Institute, S-171 76, Stockholm, Sweden. verinika.stemme@fys.ds.sll.se
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D002452:Cell Count; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D012333:RNA, Messenger