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Cardiovascular Research 1998 40(3):530-537; doi:10.1016/S0008-6363(98)00189-8
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Gene expression in rod shaped cardiac myocytes, sorted by flow cytometry

Claudius Diez and Andreas Simm*

Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) der Universität, Physiologische Chemie II, Am Hubland, D-97074 Würzburg, Germany

* Corresponding author. Tel.: +49-931-888-4128; fax: +49-931-888-4113; e-mail: simm@biozentrum.uni-wuerzburg.de

Objective: Primary cardiac myocyte cultures are usually contaminated with variable parts of different cell types, such as fibroblasts, endothelial cells and smooth muscle cells. Thus, the objective of our study was to analyse the gene expression in a pure population. Methods: To obtain an homogeneous population, cardiac myocytes from adult rats were fixed with ethanol and sorted by flow cytometry. This approach is suitable for isolating either single cells or up to several thousand cells. To measure the messenger ribonucleic acid (mRNA) expression of different genes at the level of a few rod-shaped myocytes, a cDNA library was created by polymerase chain reaction (PCR). Results: Sorting by a fluorescence-activated cell sorter (FACS) resulted in pure rod-shaped cardiac myocytes and isolated RNA from these cells is undegraded, as shown by Northern blotting. We demonstrated both the expression of housekeeping genes, such as β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as well as the myocyte-specific transcripts, {alpha}-cardiac myosin heavy chain ({alpha}-MHC) and β-MHC. Furthermore, we showed the induction of the immediately early gene c-fos at the level of ten sorted cells. Conclusions: This method allows one to study gene expression in different cell types within the heart, in tissue samples or to tackle the problem of heterogeneity within a cell population.

KEYWORDS Cardiac myocytes; Cell sorting; Single-cell RT-PCR; Gene expression; Rat; Northern blotting


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