It has recently been discovered that the expression profile of microRNA changes in the diseased heart (1, 2). Some of the dysregulated microRNAs have been shown to affect the expression of important cardiomyocyte genes, resulting in pathophysiology characteristic of the disease (3). It is likely that there are similar effects of other microRNAs that are dysregulated in heart disease. It has recently become possible to study the effects of specific microRNAs on cardiomyocyte physiology by transfecting adult rat cardiomyocytes with microRNA mimics (1). Fluorophore-tagged mimics have been used as a positive control to indicate successful transfection, although a positive control for functional activity of transfected microRNA would enhance these studies. Analysis of microRNA gene targeting has shown that miR-1 destabilises the mRNA of several target genes, with the actin-binding protein twinfilin-1 (Twf-1; PTK9) being the most strongly affected (4). Twf-1 mRNA down-regulation would therefore be a good indicator of miR-1 function. Twf-1 mRNA is known to be present in mouse heart (5), but its presence in adult rat cardiomyocytes has not been reported. We have detected Twf-1 mRNA in isolated adult rat cardiomyocytes using quantitative real-time PCR (qPCR) on an Applied Biosystems 3700 machine. Adult male Sprague-Dawley rats were anaesthetised by intraperitoneal injection of pentobarbital (100 mg/kg). Hearts were removed and perfused with Kreb’s buffer containing collagenase and hyaluronidase to isolate cardiomyocytes, which were seeded onto laminin-treated cover slips (8 mm diameter) and maintained in culture medium. The number of cells on each cover slip was estimated by counting cells in 6 fields of view. The TaqMan® Gene Expression Cells-to-CT™ Kit (Applied Biosystems) was used for cell lysis and reverse transcription. A Twf-1 TaqMan® assay (Rn01407564_g1, Applied Biosystems) was used for qPCR. Control reactions without reverse transcription (RT-) were used to estimate the maximum number of cardiomyocytes per sample that could be fully lysed using the Cells-to-CT™ protocol, as indicated by the elimination of genomic DNA by DNAse I treatment. RT- assays from ~1400 or more viable cardiomyocytes (n=4) produced amplification products, indicating incomplete cell lysis. RT- assays were negative (below amplification threshold after 40 cycles) in lysates from ~1000 or fewer viable cardiomyocytes (n=4), while assays from these lysates gave amplification products after reverse transcription (RT+). Within-sample ΔCt values for Twf-1 vs glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Rn01775763_g1, Applied Biosystems) were 3.7, 3.4 and 2.4 for 3 separate samples. These results establish the principle that quantitation of Twf-1 mRNA relative to an endogenous control gene such as GAPDH could be used as a functional assay for microRNA transfection in adult rat cardiomyocytes, using miR-1 as a positive control.