R. Preisig-muller et al., Separation of cardiomyocytes and coronary endothelial cells for cell-specific RT-PCR, AM J P-HEAR, 46(1), 1999, pp. H413-H416
Citations number
8
Categorie Soggetti
Cardiovascular & Hematology Research
Journal title
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
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.