Lee Won-Jae, Jeon Ryoung-Hoon, Jang Si-Jung, Park Ji-Sung, Lee Seung-Chan, Baregundi Subbarao Raghavendra, Lee Sung-Lim, Park Bong-Wook, King William Allan, Rho Gyu-Jin
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, 501 Jinju-daero, Jinju 660-701, Republic of Korea ; PWG Genetics Pte Ltd., 15 Tech Park Crescent, Singapore 638117.
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, 501 Jinju-daero, Jinju 660-701, Republic of Korea.
Stem Cells Int. 2015;2015:235192. doi: 10.1155/2015/235192. Epub 2015 Apr 20.
The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson's correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson's correlation revealed high correlation (r > 0.9) between NF3 and NFopt. Overall, the present study showed that HMBS, YWHAZ, SDHA, and TBP are suitable reference genes for qRT-PCR in porcine MSCs.
鉴定稳定的内参基因是确保基因表达准确验证的前提条件，然而对于猪间充质干细胞（MSCs）的稳定内参基因却知之甚少。因此，本研究旨在评估来自骨髓（BMSCs）、脂肪（AMSCs）和皮肤（SMSCs）的猪MSCs及其体外分化为脂肪细胞、骨细胞和软骨细胞等间充质谱系的细胞中内参基因的稳定性。通过qRT-PCR研究了12个常用内参基因的阈值循环（Ct）值。利用geNorm软件分析候选内参基因的Ct值，以阐明无论实验条件如何均能稳定表达。因此，应用Pearson相关性分析来确定三个最稳定的内参基因（NF3）与最佳内参基因数量（NFopt）之间的相关性。通过geNorm分析评估不同实验条件下内参基因的稳定性时，未分化的MSCs以及每种向间充质谱系的分化状态显示出略有不同的结果，但在稳定程度高低排序方面具有相似模式。此外，Pearson相关性分析显示NF3与NFopt之间具有高度相关性（r > 0.9）。总体而言，本研究表明HMBS、YWHAZ、SDHA和TBP是猪MSCs中qRT-PCR合适的内参基因。
