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 isolated from guinea pig hearts by collagenase digestion. In the diluted cell suspension, single cardiomyocytes and capillary fragments containing 6-15 endothelial cells could be identified morphologically. A simple "cell picker" was constructed using a polyethylene pipette with a tip diameter of approximately 150 micrometers that was attached to a micromanipulator and connected to an electric 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. Cardiomyocytes (800-1,000) or capillary fragments (150) were picked under visual control using an inverted microscope. The cells were transferred to a reaction tube for RNA extraction, reverse transcription (RT), and DNA amplification (RT-PCR) with gene-specific and intron-spanning primers. All PCR products were verified by sequencing. Troponin T and endothelin-1 were found to be specific markers for guinea-pig cardiac muscle cells and coronary endothelial cells, respectively.
Preisig-Müller
R
R
Institut für Normale und Pathologische Physiologie, Universität Marburg, D-35037 Marburg, Germany. preisigm@mailer.uni-marburg.de
Mederos y Schnitzler
M
M
Derst
C
C
Daut
J
J
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D003331:Coronary Vessels / Q000166:cytology* / Q000378:metabolism; D019332:Endothelin-1 / Q000096:biosynthesis; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D006168:Guinea Pigs; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014336:Troponin / Q000096:biosynthesis
D019332:Endothelin-1; D014336:Troponin