Department of Pediatrics, University Hospital Carl Gustav Carus, TU Dresden, Fetscherstraße 74, 01307, Dresden, Germany.
Experimental Trauma Surgery, Justus-Liebig University, Aulweg 128, 35392, Giessen, Germany.
BMC Genomics. 2017 Dec 19;18(1):975. doi: 10.1186/s12864-017-4356-4.
In 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.
Twelve 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.
We 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.
This 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.
为了更好地理解骨质疏松症的多因素性质，人们利用动物模型并将其与健康对照组进行比较。雌性绵羊已被广泛建立为去卵巢、低钙和维生素 D 饮食、应用类固醇或这些治疗方法组合引起的骨质疏松症的模型。可以通过应用实时定量聚合酶链反应 (RT-qPCR) 进行转录研究。RT-qPCR 估计目标基因相对于参考基因的 mRNA 水平。选择的一组参考基因不应在实验条件下显示变化。目前，没有一种标准参考基因适用于所有组织类型和实验条件。研究绵羊参考基因的研究很少，只有一项研究描述了下颌骨的稳定参考。然而，这种类型的骨骼与大多数骨质疏松性骨折发生的小梁骨不同。本研究旨在确定一组参考基因，用于相对定量绵羊脊柱骨和绵羊体外分化间充质基质细胞 (MSC) 的转录活性，以实现可靠的可比性。
选择了 12 个属于不同功能类别的候选参考基因，并分别测量了培养的 ovMSCs（n=18）和绵羊骨样本（n=16）中的表达。使用 RefFinder 对候选基因进行排名。
我们确定 B2M、GAPDH、RPL19 和 YWHAZ 是这些绵羊样本转录分析中 RT-qPCR 结果标准化的最佳参考基因组合。
本研究证明了在绵羊中应用一组参考基因进行 RT-qPCR 分析的重要性。基于我们的数据，我们建议在绵羊骨或体外与成骨分化的绵羊 MSC 进行实验中，使用四个鉴定的参考基因进行基因表达研究的相对定量。
