Usarek Ewa, Barańczyk-Kuźma Anna, Kaźmierczak Beata, Gajewska Beata, Kuźma-Kozakiewicz Magdalena
Department of Biochemistry, Medical University of Warsaw, Warsaw, Poland.
Neurodegenerative Diseases Research Group, Medical University of Warsaw, Warsaw, Poland.
PLoS One. 2017 Mar 22;12(3):e0174317. doi: 10.1371/journal.pone.0174317. eCollection 2017.
Quantitative polymerase chain reaction (qPCR) is the most specific and reliable method for determination of mRNA gene expression. Crucial point for its accurate normalization is the choice of appropriate internal control genes (ICGs). In the present work we determined and compare the expression of eight commonly used ICGs in lymphocytes from 26 patients with amyotrophic lateral sclerosis (ALS) and 30 control subjects. Peripheral blood mononuclear cells (PBMCs) before and after immortalization by EBV transfection (lymphoblast cell lines-LCLs) were used for qPCR analysis. LCLs were studied before and after liquid nitrogen cryopreservation and culturing (groups LCL1 and LCL2, respectively). qPCR data of 8 ICGs expression was analyzed by BestKeeper, NormFinder and geNorm methods. All studied genes (18SRNA, ACTB, B2M, GUSB,GAPDH, HPRT1, MT-ATP6 and RPS17) were expressed in PBMCs, whereas only first four in LCLs. LCLs cryopreservation had no effect on ICGs expression. Comprehensive ranking indicated RPS17 with MT-ATP6 as the best ICGs for qPCR in PBMCs of control and ALS subjects, and RPS17 with 18RNA or MT-ATP6 in LCLs from ALS. In PBMCs 18RNA shouldn't be used as ICG.
定量聚合酶链反应（qPCR）是测定mRNA基因表达最特异、最可靠的方法。其准确标准化的关键点是选择合适的内参基因（ICG）。在本研究中，我们测定并比较了26例肌萎缩侧索硬化症（ALS）患者和30例对照者淋巴细胞中8种常用ICG的表达。通过EBV转染永生化前后的外周血单个核细胞（PBMC）（淋巴母细胞系-LCL）用于qPCR分析。分别在液氮冷冻保存和培养前后对LCL进行研究（分别为LCL1和LCL2组）。采用BestKeeper、NormFinder和geNorm方法分析8种ICG表达的qPCR数据。所有研究基因（18SRNA、ACTB、B2M、GUSB、GAPDH、HPRT1、MT-ATP6和RPS17）均在PBMC中表达，而在LCL中只有前四种表达。LCL冷冻保存对ICG表达无影响。综合排名表明，RPS17与MT-ATP6是对照和ALS受试者PBMC中qPCR的最佳ICG，而在ALS患者的LCL中，RPS17与18RNA或MT-ATP6是最佳ICG。在PBMC中，18RNA不应用作ICG。
