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We established a short tandem repeat (STR) profile for H9c2 that is useful to confirm the identity and stability of the cell line. Additionally, we prepared H9c2 metaphase chromosomes and performed karyotyping and molecular cytogenetics to further investigate chromosomal characteristics. The genetic analysis showed that H9c2 cells exhibit chromosomal instability, which may impact experimental reproducibility and data interpretation. Next-generation sequencing (NGS) was performed to analyze the transcriptome, revealing gene expression patterns relevant to cardiac biology. Western blot analysis further validated the expression levels of selected cardiac genes identified through NGS. Additionally, Phalloidin staining was used to visualize cytoskeletal organization, highlighting the morphological features of these cardiac myoblasts. Our findings collectively support that H9c2 cells are a reliable model for studying cardiac myoblast biology, despite some genetic alterations identified resembling sarcoma cells. The list of genes identified through NGS analysis, coupled with our comprehensive genetic analysis, will serve as a valuable resource for future studies utilizing this cell line in cardiovascular medicine.<\/jats:p>","DOI":"10.3390\/cells14070502","type":"journal-article","created":{"date-parts":[[2025,3,28]],"date-time":"2025-03-28T06:06:35Z","timestamp":1743141995000},"page":"502","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":22,"title":["Genetic and Molecular Characterization of H9c2 Rat Myoblast Cell Line"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1672-3054","authenticated-orcid":false,"given":"Thomas","family":"Liehr","sequence":"first","affiliation":[{"name":"Institute of Human Genetics, Jena University Hospital, Friedrich Schiller University, D-07747 Jena, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stefanie","family":"Kankel","sequence":"additional","affiliation":[{"name":"Institute of Human Genetics, Jena University Hospital, Friedrich Schiller University, D-07747 Jena, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katharina S.","family":"Hardt","sequence":"additional","affiliation":[{"name":"Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, D-52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0627-9228","authenticated-orcid":false,"given":"Eva M.","family":"Buhl","sequence":"additional","affiliation":[{"name":"Electron Microscopy Facility, Institute of Pathology, RWTH University Hospital Aachen, D-52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3053-6984","authenticated-orcid":false,"given":"Heidi","family":"Noels","sequence":"additional","affiliation":[{"name":"Institute for Molecular Cardiovascular Research (IMCAR), RWTH University Hospital Aachen, D-52074 Aachen, Germany"},{"name":"Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, 6211 Maastricht, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Diandra T.","family":"Keller","sequence":"additional","affiliation":[{"name":"Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, D-52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2265-3726","authenticated-orcid":false,"given":"Sarah K.","family":"Schr\u00f6der-Lange","sequence":"additional","affiliation":[{"name":"Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, D-52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3888-0931","authenticated-orcid":false,"given":"Ralf","family":"Weiskirchen","sequence":"additional","affiliation":[{"name":"Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, D-52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1016\/0014-4827(76)90447-X","article-title":"Properties of a clonal muscle cell line from rat heart","volume":"98","author":"Kimes","year":"1976","journal-title":"Exp. 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