Sood Tanushri Jerath, Lagah Swati Viviyan, Sharma Ankita, Singla Suresh Kumar, Mukesh Manishi, Chauhan Manmohan Singh, Manik Radheysham, Palta Prabhat
1 Animal Biotechnology Center, ICAR-National Dairy Research Institute , Karnal, India .
2 ICAR-National Bureau of Animal Genetic Resources , Karnal, India .
Cell Reprogram. 2017 Oct;19(5):302-310. doi: 10.1089/cell.2017.0010. Epub 2017 Sep 7.
We evaluated the suitability of 10 candidate internal control genes (ICGs), belonging to different functional classes, namely ACTB, EEF1A1, GAPDH, HPRT1, HMBS, RPS15, RPS18, RPS23, SDHA, and UBC for normalizing the real-time quantitative polymerase chain reaction (qPCR) data of blastocyst-stage buffalo embryos produced by hand-made cloning and in vitro fertilization (IVF). Total RNA was isolated from three pools, each of cloned and IVF blastocysts (n = 50/pool) for cDNA synthesis. Two different statistical algorithms geNorm and NormFinder were used for evaluating the stability of these genes. Based on gene stability measure (M value) and pairwise variation (V value), calculated by geNorm analysis, the most stable ICGs were RPS15, HPRT1, and ACTB for cloned blastocysts, HMBS, UBC, and HPRT1 for IVF blastocysts and RPS15, GAPDH, and HPRT1 for both the embryo types analyzed together. RPS18 was the least stable gene for both cloned and IVF blastocysts. Following NormFinder analysis, the order of stability was RPS15 = HPRT1>GAPDH for cloned blastocysts, HMBS = UBC>RPS23 for IVF blastocysts, and HPRT1>GAPDH>RPS15 for cloned and IVF blastocysts together. These results suggest that despite overlapping of the three most stable ICGs between cloned and IVF blastocysts, the panel of ICGs selected for normalization of qPCR data of cloned and IVF blastocyst-stage embryos should be different.
我们评估了10个候选内参基因（ICGs）的适用性，这些基因属于不同的功能类别，即肌动蛋白β（ACTB）、真核生物延伸因子1α1（EEF1A1）、甘油醛-3-磷酸脱氢酶（GAPDH）、次黄嘌呤磷酸核糖转移酶1（HPRT1）、羟甲基丁二酰辅酶A合成酶（HMBS）、核糖体蛋白S15（RPS15）、核糖体蛋白S18（RPS18）、核糖体蛋白S23（RPS23）、琥珀酸脱氢酶复合物亚基A（SDHA）和泛素羧基末端水解酶L1（UBC），用于对通过手工克隆和体外受精（IVF）产生的囊胚期水牛胚胎的实时定量聚合酶链反应（qPCR）数据进行标准化。从三个样本库中分离总RNA，每个样本库包含克隆囊胚和IVF囊胚（每个样本库n = 50）用于cDNA合成。使用两种不同的统计算法geNorm和NormFinder来评估这些基因的稳定性。根据geNorm分析计算的基因稳定性度量（M值）和成对变异（V值），对于克隆囊胚，最稳定的ICGs是RPS15、HPRT1和ACTB；对于IVF囊胚，是HMBS、UBC和HPRT1；对于一起分析的两种胚胎类型，是RPS15、GAPDH和HPRT1。RPS18是克隆囊胚和IVF囊胚中最不稳定的基因。经过NormFinder分析，克隆囊胚的稳定性顺序为RPS15 = HPRT1>GAPDH，IVF囊胚为HMBS = UBC>RPS23，克隆囊胚和IVF囊胚一起为HPRT1>GAPDH>RPS15。这些结果表明，尽管克隆囊胚和IVF囊胚之间三个最稳定的ICGs存在重叠，但选择用于标准化克隆囊胚和IVF囊胚期胚胎qPCR数据的ICGs组合应该不同。
