Abstract
BackgroundIn order to better understand the multifactorial nature of osteoporosis, animal models are utilized and compared to healthy controls. Female sheep are well established as a model for osteoporosis induced by ovariectomy, calcium and vitamin D low diet, application of steroids, or a combination of these treatments. Transcriptional studies can be performed by applying quantitative real time PCR (RT-qPCR). RT-qPCR estimates mRNA-levels of target genes in relation to reference genes. A chosen set of reference genes should not show variation under experimental conditions. Currently, no standard reference genes are accepted for all tissue types and experimental conditions. Studies examining reference genes for sheep are rare and only one study described stable reference in mandibular bone. However, this type of bone differs from trabecular bone where most osteoporotic fractures occur. The present study aimed at identifying a set of reference genes for relative quantification of transcriptional activity of ovine spine bone and ovine in vitro differentiated mesenchymal stromal cells (MSC) for reliable comparability.MethodsTwelve candidate reference genes belonging to different functional classes were selected and their expression was measured from cultured ovMSCs (n = 18) and ovine bone samples (n = 16), respectively. RefFinder was used to rank the candidate genes.ResultsWe identified B2M, GAPDH, RPL19 and YWHAZ as the best combination of reference genes for normalization of RT-qPCR results for transcriptional analyses of these ovine samples.ConclusionThis study demonstrates the importance of applying a set of reference genes for RT-qPCR analysis in sheep. Based on our data we recommend using four identified reference genes for relative quantification of gene expression studies in ovine bone or for in vitro experiments with osteogenically differentiated ovine MSCs.
Highlights
In order to better understand the multifactorial nature of osteoporosis, animal models are utilized and compared to healthy controls
In order to better understand the multifactorial nature of osteoporosis, small and large animal models are utilized upon the recommendations of the Food and Drug Administration (FDA)
Ubiquitous expression of tested reference genes was considered by BioGPS sheep gene expression atlas dataset [21]
Summary
In order to better understand the multifactorial nature of osteoporosis, animal models are utilized and compared to healthy controls. The present study aimed at identifying a set of reference genes for relative quantification of transcriptional activity of ovine spine bone and ovine in vitro differentiated mesenchymal stromal cells (MSC) for reliable comparability. Osteoporosis in sheep is induced by ovariectomy, Besides recent advances, e.g. in terms of using RNAsequencing or DNA-microarrays, quantitative real time PCR (RT-qPCR) is still used for quantifying mRNA to reflect relative expression of genes of interest (GOI) or to evaluate results obtained from RNA-sequencing. This is a powerful tool because of its high sensitivity, specificity, and low cost.
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