Utility of the housekeeping genes 18S rRNA, β-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes
Utility of the housekeeping genes 18S rRNA, β-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes
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DOI：
10.1111/j.0300-9475.2004.01440.x
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发表时间：
2004-06-01
影响因子：
3.7
通讯作者：
Hammarström, ML
中科院分区：
文献类型：
--
作者：
Bas, A;Forsberg, G;Hammarström, ML
研究了18 S rRNA、β-肌动蛋白mRNA和甘油醛-3-磷酸脱氢酶（GAPDH）mRNA作为细胞数指标用于不同活化阶段T淋巴细胞基因表达分析的标准化时的准确性。采用实时定量逆转录-聚合酶链反应（RT-PCR）测定静息人外周血单个核细胞（PBMC）、肠淋巴细胞和多克隆T细胞活化的PBMC的18 S rRNA、β-肌动蛋白mRNA、GAPDH mRNA和6种细胞因子mRNA的表达水平。活化和静息PBMC和肠淋巴细胞中的18 S rRNA水平基本相同，而β-肌动蛋白和GAPDH mRNA的水平在活化后显著波动。当研究分离的γ δ TCR（+）、CD 4（+）和CD 8（+）亚群时，18 S rRNA水平在活化21 h后保持不变，但在96 h后略有增加。相比之下，在活化后，这些细胞群中GAPDH mRNA/细胞增加30-70倍。细胞因子分析显示，仅归一化至18 S rRNA得到的结果令人满意地反映了每个细胞的mRNA表达水平。总之，18 S rRNA是最稳定的管家基因，因此在人T淋巴细胞mRNA表达水平的比较分析中，18 S rRNA具有上级标准化优势。
The accuracy of 18S rRNA, beta-actin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as indicators of cell number when used for normalization in gene expression analysis of T lymphocytes at different activation stages was investigated. Quantitative real-time reverse transcriptase-polymerase chain reaction was used to determine the expression level of 18S rRNA, beta-actin mRNA, GAPDH mRNA and mRNA for six cytokines in carefully counted samples of resting human peripheral blood mononuclear cells (PBMCs), intestinal lymphocytes and PBMCs subjected to polyclonal T-cell activation. The 18S rRNA level in activated and resting PBMCs and intestinal lymphocytes was essentially the same, while the levels of beta-actin and GAPDH mRNAs fluctuated markedly upon activation. When isolated gammadeltaTCR(+), CD4(+) and CD8(+) subpopulations were studied, 18S rRNA levels remained unchanged after 21 h of activation but increased slightly after 96 h. In contrast, there was a 30-70-fold increase of GAPDH mRNA/cell in these cell populations upon activation. Cytokine analysis revealed that only normalization to 18S rRNA gave a result that satisfactorily reflected their mRNA expression levels per cell. In conclusion, 18S rRNA was the most stable housekeeping gene and hence superior for normalization in comparative analyses of mRNA expression levels in human T lymphocytes.