Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood.
Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood.
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DOI：
10.1186/1755-8794-2-49
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发表时间：
2009-08-05
影响因子：
2.7
通讯作者：
Sharp FR
中科院分区：
文献类型：
--
作者：
Stamova BS;Apperson M;Walker WL;Tian Y;Xu H;Adamczy P;Zhan X;Liu DZ;Ander BP;Liao IH;Gregg JP;Turner RJ;Jickling G;Lit L;Sharp FR
基因表达研究需要适当的规范化方法。其中一种方法使用稳定表达的内参基因。由于合适的内参基因似乎对每个组织都是独一无二的，我们已经确定了一组人类血液中最稳定表达的基因，可用于标准化。全基因组Affymetrix Human 2.0 Plus阵列检测了526个年龄在2到78岁之间的男性和女性样本，包括对照组和患有图雷特综合征、中风、偏头痛、肌肉萎缩症和自闭症的患者。鉴定出100个表达最稳定、表达水平范围广的基因。为了验证最佳候选基因，我们对10个基因（TRAP1、DECR1、FPGS、FARP1、MAPRE2、PEX16、GINS2、CRY2、CSNK1G2和A4GALT）、4个常用的内参基因（GAPDH、ACTB、B2M和HMBS）和PPIB进行了定量RT-PCR，这些基因之前报道在血液中稳定表达。使用GeNorm和NormFinder算法进行表达稳定性和排序分析。根据内参基因的表达稳定性对其进行排序，使用GeNorm计算归一化所需的最小基因数量表明，在人全血RNA表达研究中，准确归一化所需的最少、最稳定表达的基因是TRAP1、FPGS、DECR1和PPIB的组合。我们使用另一种算法（NormFinder）确定了最佳候选控制基因的排名。本研究中鉴定的内参基因在2至78岁的患有多种疾病的男女全血中稳定表达。重要的是，它们在细胞内也有不同的功能，因此应该相互独立地表达。这些基因可作为人体生理、代谢和疾病的微阵列和RT-PCR全血研究的规范化基因。
Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT), 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS) and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms. Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder). The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.
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影响因子：
--
作者：
Silver N;Best S;Jiang J;Thein SL
通讯作者：
Thein SL
影响因子：
3.7
作者：
Roge, R.;Thorsen, J.;Carstens, J.
通讯作者：
Carstens, J.
影响因子：
--
作者：
Jung M;Ramankulov A;Roigas J;Johannsen M;Ringsdorf M;Kristiansen G;Jung K
通讯作者：
Jung K
影响因子：
15.1
作者：
Enstrom, Amanda M.;Lit, Lisa;Onore, Charity E.;Gregg, Jeff P.;Hansen, Robin L.;Pessah, Isaac N.;Hertz-Picciotto, Irva;Van de Water, Judy A.;Sharp, Frank R.;Ashwood, Paul
通讯作者：
Ashwood, Paul
DOI：
10.1002/ajmg.b.30550
发表时间：
2007-10-05
影响因子：
2.8
作者：
Lit, Lisa;Gilbert, Donald L.;Sharp, Frank R.
通讯作者：
Sharp, Frank R.