# Identification of Reference Gene for Quantitative Gene Expression in Early-Term and Late-Term Cultured Canine Fibroblasts Derived from Ear Skin

## TL;DR

This study identifies the most stable reference genes for measuring gene expression in canine skin fibroblasts at different culture stages.

## Contribution

HPRT1, YWHAZ, and GUSB are shown to be the most stable reference genes for RT-qPCR in both early- and late-passage canine fibroblasts.

## Key findings

- Early-passage fibroblasts showed shorter doubling times and lower β-galactosidase activity compared to late-passage cells.
- HPRT1, YWHAZ, and GUSB were identified as the most stable reference genes across three algorithms.
- Using stable reference genes revealed significant differences in Vimentin expression not seen with unstable genes.

## Abstract

This study aimed to identify stable reference genes in early-passage and late-passage cultured canine skin fibroblasts. The early-passage fibroblasts retained their spindle-shaped morphology, exhibited a short doubling time, and had low β-galactosidase activity. In contrast, the late-passage fibroblasts displayed an elongated morphology, a prolonged doubling time, and elevated β-galactosidase activity. To assess the stability of the reference genes, the Ct values obtained using qRT-PCR were analyzed using three algorithms: geNorm, NormFinder, and BestKeeper. As a result, HPRT1, YWHAZ, and GUSB were identified as the most stable reference genes across all three algorithms in canine skin fibroblasts. When comparing early-passage to late-passage fibroblasts, the normalization of Vimentin expression using both stable and unstable reference genes showed a decrease in late-passage cells. Although the use of less stable reference genes did not result in a significant difference in Vimentin expression, the use of stable reference genes revealed a significant difference. This study provides a foundation for the further application of RT-qPCR in the gene expression analysis of long-term expanded canine skin fibroblasts.

Fibroblasts are cells that reside within the fibrous or loose connective tissues of most mammalian organs. For research purposes, fibroblasts are often subjected to long-term culture under defined conditions, during which their properties can significantly change. It is essential to understand and document these changes to obtain reliable outcomes. For the quantification of specific gene expressions, the most reliable and widely used technique is quantitative real-time polymerase chain reaction (qRT-PCR). Here, we assessed the impact of a reference gene’s stability on a qRT-PCR analysis of long-term cultured canine skin fibroblasts. After successfully isolating the fibroblasts from canine skin tissues, they were cultured and evaluated for proliferation and β-galactosidase activity at different passage numbers. With extended culture, the fibroblasts showed a long doubling time and elevated β-galactosidase activity. Using three widely used algorithms, geNorm, Normfinder, and Bestkeeper, we identified HPRT1, YWHAZ, and GUSB as the most stable reference genes for both early- and late-passage fibroblasts. Conventional reference genes such as GAPDH were found to be less stable than those genes. The normalization of Vimentin by the stable genes showed statistical differences, whereas normalization by an unstable gene did not. Collectively, this study indicates that using stable reference genes is essential for accurately and reliably measuring gene expression in both early- and late-passage fibroblasts. These findings provide valuable insights into internal controls for gene expression studies and are expected to be utilized for analyzing gene expression patterns in molecular biology research.