Determination of novel reference genes for improving gene expression data normalization in selected canine reproductive tissues – a multistudy analysis

Abstract

Background Real time RT-PCR (qPCR) is a useful and powerful tool for quantitative measurement of gene expression. The proper choice of internal standards such as reference genes is crucial for correct data evaluation. In female dogs, as in other species, the reproductive tract is continuously undergoing hormonal and cycle stage-dependent morphological changes, which are associated with altered gene expression. However, there have been few attempts published so far targeted to the dog aimed at determining optimal reference genes for the reproductive organs. Most of these approaches relied on genes previously described in other species. Large-scale transcriptome-based experiments are promising tools for defining potential candidate reference genes, but were never considered in this context in canine research. Results Here, using available microarray and RNA-seq datasets derived from reproductive organs (corpus luteum, placenta, healthy and diseased uteri) of dogs, we have performed multistudy analysis to identify the most stably expressed genes for expression studies, in each tissue separately and collectively for different tissues. The stability of newly identified reference genes (EIF4H, KDELR2, KDM4A and PTK2) has been determined and ranked relative to previously used reference genes, i.e., GAPDH, β-actin and cyclophillin A/PPIA, using RefFinder and NormFinder algorithms. Finally, expression of selected target genes (luteal IL-1b and MHCII, placental COX2 and VEGFA, and uterine IGF2 and LHR) was re-evaluated and normalized. All proposed candidate reference genes were more stable, ranked higher and introduced less variation than previously used genes. Conclusions Based on our analyses, we recommend applying KDM4A and PTK2 for normalization of gene expression in the canine CL and placenta. The inclusion of a third reference gene, EIF4H, is suggested for healthy uteri. With this, the interpretation of qPCR data will be more reliable, allowing better understanding of canine reproductive physiology.

Molecular studies on oestrogen α and progesterone receptors and histomorphometric analysis of canine uteri following aglepristone treatment

“…The reference gene also relates to sample types, RNA preparation and processing, and reproductive tissues undergoing hormonal changes (Nowak et al, 2020). Recently, new housekeeping genes for the evaluation of canine reproductive tissue were recommended by Nowak et al (2020) Based on the pharmacokinetics of aglepristone, two injections of 10 mg/kg/day, 24 hr apart, the Cmax, the Tmax and the mean residence time (MRT) were 280 ng/ml, 2.7 days and 6 days, respectively (Plumb, 2011). For a single injection of the 5 and 20 mg/kg, the MRT were 4.5 and 6.5 days, respectively (Virbac Animal Health, 2010).…”

Antigestagens Mediate the Expression of Decidualization Markers, Extracellular Matrix Factors and Connexin 43 in Decidualized Dog Uterine Stromal (DUS) Cells

“…The efficiency of probes was evaluated to ensure approximately 100%, as previously described [ 29 , 31 ]. Relative gene expression was quantified with the ΔΔCt method, using KDM4A, EIF4H and PTK2, reported to be stably expressed in the canine uterus [ 32 ], as reference genes.…”

Involvement of Oxytocin and Progesterone Receptor Expression in the Etiology of Canine Uterine Inertia

“…cDNA was stored at −20 °C until further use. Specific primer sets for both OXTR and PGR for quantitative real-time polymerase chain reaction (RT-qPCR) were purchased from TAG Copenhagen (TAG Copenhagen A/S, Copenhagen, Denmark) and primers for the reference genes glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), protein tyrosine kinase 2 ( PTK2 ), eukaryotic translation initiation factor 4H ( EIF4H ), and lysine-specific demethylase 4A ( KDM4A ) [ 75 ] from Microsynth (Microsynth AG; Balgach, Switzerland) ( Table 1 ). The specificity of the primers was checked with BLAST ( , accessed on 3 August 2021).…”