Commonly used reference genes are actively regulated in in vitro stimulated lymphocytes
Commonly used reference genes are actively regulated in in vitro stimulated lymphocytes
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DOI：
10.1111/j.1365-3083.2006.01879.x
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发表时间：
2007-02-01
影响因子：
3.7
通讯作者：
Carstens, J.
中科院分区：
文献类型：
--
作者：
Roge, R.;Thorsen, J.;Carstens, J.
定量聚合酶链反应（Q-PCR）研究表明，在同种异体肾移植急性排斥发作期间，尿液沉积物中细胞毒素基因的表达增加。为了弥补初始样本量和cDNA制备的差异，标准Q-PCR实验涉及到标准化的参考基因。虽然内参基因的稳定表达是任何Q-PCR分析的先决条件，但常用的内参基因在组织和各种刺激下表现出不同的表达。在这项研究中，研究了混合淋巴细胞反应中几种内参基因的细胞表达，作为同种异体t淋巴细胞活化和急性排斥反应期间基因表达的模型。使用Q-PCR定量表达基因，通过DNA定量归一化为细胞计数，并使用流式细胞术校正细胞多倍体。检测的内参基因为18S rRNA、β -肌动蛋白（ACTB）、羟甲基二烷合成酶（HMBS）、次黄嘌呤磷酸核糖基转移酶（HPRT1）和肽基丙氨酸异构酶B （PPIB）。本研究还检测了两个新的t淋巴细胞特异性内参基因：CD3E和CD8B。HMBS和HPRT分别上调18.8倍和7.4倍，ACTB上调5.3倍，PPIB上调3.2倍，而18S rRNA保持稳定表达。t淋巴细胞特异性参考基因CD3E保持稳定，而CD8B上调2.3倍。综上所述，几种常用的内参基因在同种异体t淋巴细胞活化过程中受到积极调控。此外，我们引入了两个稳定的t淋巴细胞特异性内参基因，它们可能有助于尿液沉积物中t淋巴细胞特异性细胞毒素基因的Q-PCR分析，因为它们克服了来自与诊断无关的细胞的内参基因mRNA的贡献。
Quantitative polymerase chain reaction (Q-PCR) studies of urine sediments have demonstrated an increased expression of cytotoxin genes during episodes of acute rejection of renal allografts. To compensate for differences in initial sample size and cDNA preparation, standard Q-PCR experiments involve normalization to a reference gene. Although stable expression of reference genes is a prerequisite for any Q-PCR analysis, commonly used reference genes have demonstrated a varying expression across tissues and various stimuli. In this study, cellular expression of several reference genes was investigated in a mixed lymphocyte reaction as a model of gene expression during alloreactive T-lymphocyte activation and acute rejection. Gene expression was quantified using Q-PCR, normalized to cell counts obtained by DNA quantification and corrected for cell polyploidy using flow cytometry. Examined reference genes were 18S rRNA, beta-actin (ACTB), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT1) and peptidylprolyle isomerase B (PPIB). This study also examined two novel T-lymphocyte-specific reference genes: CD3E and CD8B. HMBS and HPRT were 18.8- and 7.4-fold upregulated, respectively, ACTB was 5.3-fold upregulated, PPIB was 3.2-fold upregulated while 18S rRNA remained stably expressed. The T-lymphocyte-specific reference gene CD3E remained stable while CD8B was upregulated 2.3-fold. In conclusion, several commonly used reference genes were actively regulated during alloreactive T-lymphocyte activation. Additionally, we introduce two stable T-lymphocyte-specific reference genes that might be useful in a Q-PCR analysis of T-lymphocyte-specific cytotoxin genes in urine sediments, as they overcome the contribution of reference gene mRNA from cells irrelevant for diagnosis.