Separation of cardiomyocytes and coronary endothelial cells for cell-specific RT-PCR

A simple method for analyzing the differential gene expression of coronary endothelial cells and cardiac muscle cells was developed. Cells were isolat ed from guinea pig hearts by collagenase digestion. In the diluted cell sus pension, single cardiomyocytes and capillary fragments containing 6-15 endo thelial cells could be identified morphologically. A simple "cell picker" w as constructed using a polyethylene pipette with a tip diameter of similar to 150 mu m that was attached to a micromanipulator and connected to an ele ctric miniature valve. Intermittent suction pulses (1- to 2-cm water column ) were applied by opening the valve for 100-200 ms at 1-s intervals. Cardio myocytes (800-1,000) or capillary fragments (150) were picked under visual control using an inverted microscope. The cells were transferred to a react ion tube for RNA extraction, reverse transcription (RT), and DNA amplificat ion (RT-PCR) with gene-specific and intron-spanning primers. All PCR produc ts were verified by sequencing. Troponin T and endothelin-l were found to b e specific markers for guinea-pig cardiac muscle cells and coronary endothe lial cells, respectively.