Evaluation of gamma-actin, beta-actin, GAPDH, and 18S as reference genes for qRT-PCR using blood samples in canine mammary research

Abstract

Mammary tumours are the most frequent group of neoplasia in female dogs. Tumorigenesis is associated with gene expression changes in a wide variety of genes. For this reason, real-time quantitative PCR (qRT-PCR) is used in routine diagnostic procedures in clinical practice due to its the specificity, sensitivity, simplicity, and high performance. qRT-PCR is also widely used to measure the expression of target genes compared to reference genes in several tissues. We collected blood samples from healthy female dogs and females with canine mammary cancer in Manizales, Colombia between June 2018 and January 2019, and mRNA was isolated from each sample for cDNA synthesis. qRT-PCR-based expression assays were performed using primers designed for gamma-actin, beta-actin, GAPDH, and 18S genes. We calculated the amplification efficiency, specificity, and stability using geNorm, NormFinder, BestKeeper, and the ΔCt comparative method. We obtained linear regressions to verify constant gene expression and conducted an ANOVA to detect expression differences regarding Ct values and healthy vs. ill conditions. We found stability for primers 18S-1, GAPDH-1, GAPDH-NM, and Gamma-actin-1 (in increasing order). Furthermore, these genes showed constant expression levels in patients (R2>0.80). We report novel primers for gamma-actin and GAPDH, which proved to be efficient endogenous control genes for qRT-PCR applications in blood tissue. These primers are useful for gene expression research in canine mammary cancer.