Abstract
BackgroundReal 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.ResultsHere, 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.ConclusionsBased 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.
Highlights
Real time Reverse transcription (RT)-PCR is a useful and powerful tool for quantitative measurement of gene expression
Tissue-specific candidates for reference genes Applying the criteria described in Methods, we identified 1649 potential candidates for reference genes for placenta (Fig. 1a), 430 for corpora lutea (CL) (Fig. 1b) and 18 for all uterine samples (Fig. 1c)
Four genes from different functional categories were selected for further validation and included: eukaryotic translation initiation factor 4H (EIF4H), endoplasmic reticulum lumen protein-retaining receptor 2 (KDELR2), lysine-specific demethylase 4A (KDM4A) and protein tyrosine kinase 2 (PTK2)
Summary
Real time RT-PCR (qPCR) is a useful and powerful tool for quantitative measurement of gene expression. 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. The most often applied normalization strategy is the use of internal controls, i.e., reference genes [6] These genes are supposed to be stably expressed in the examined tissues and among experimental groups, and, if properly validated, allow controlling for errors that might be introduced during the procedure. It has to be emphasized that proper normalization is of utmost importance since it allows overcoming of pitfalls related to sample preparation and processing These include, i.a., RNA extraction technique, sample quality, applied DNase treatment and RT method. Poor validation of reference genes can lead to overlooking of discrete changes in gene expression, thereby generating false data, or can result in misunderstanding of underlying biological processes [3]
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