Study Protocol
Neonatal rat fibroblast isolation, culture, differentiation and determination of microRNA effects in transformation
Abstract
Background: Cardiac fibrosis is a significant global health problem associated with nearly all forms of cardiovascular disease, lacking effective treatments. Upon myocardial injury, cardiac fibroblasts transform to an activated myofibroblast phenotype and contribute to cardiac fibrosis. MicroRNAs (miRNAs) are non-coding RNAs that play essential roles in modulating gene expression in fibrotic heart, thus targeting miRNAs could be a promising direction in cardiac fibrosis therapies.
Methods: This paper provides detailed protocols for neonatal rat fibroblast isolation, culture and differentiation upon serum or serum-free protocols [induced by transforming growth factor-β (TGF-β) and angiotensin II (AngII)]. Furthermore, we also show simple methods for miRNA transfection and immunostaining of α-smooth muscle actin (α-SMA) to determine miRNA effects in transformation.
Discussion: This paper will offer a powerful tool to target this cell type and its pathological contribution to disease progression.
Methods: This paper provides detailed protocols for neonatal rat fibroblast isolation, culture and differentiation upon serum or serum-free protocols [induced by transforming growth factor-β (TGF-β) and angiotensin II (AngII)]. Furthermore, we also show simple methods for miRNA transfection and immunostaining of α-smooth muscle actin (α-SMA) to determine miRNA effects in transformation.
Discussion: This paper will offer a powerful tool to target this cell type and its pathological contribution to disease progression.
