β-actin -: an unsuitable internal control for RT-PCR
β-actin -: an unsuitable internal control for RT-PCR
复制标题
DOI：
10.1006/mcpr.2001.0376
复制
发表时间：
2001-10-01
影响因子：
3.3
通讯作者：
Griffiths, LR
中科院分区：
文献类型：
--
作者：
Selvey, S;Thompson, EW;Griffiths, LR
尽管有报道证实了细胞周期依赖性基因表达和许多研究描述了β -肌动蛋白也受到调节的特定情况，但β -肌动蛋白的mRNA仍然是广泛使用的内控基因。利用差异逆转录聚合酶链反应（RT-PCR），我们在这里报告了matrigel对β -肌动蛋白的剂量依赖性抑制。这是通过与靶基因膜1型基质金属蛋白酶（MT1-MMP）非常温和的抑制进行比较而检测到的，使用竞争性RT-PCR对MT1-MMP表达的类似发现独立证实了这一结果。此外，管家基因18 Svedberg单位(S) rRNA的RT-PCR显示出良好的一致性，可重复性和矩阵处理的非调控性。我们的结论是-肌动蛋白受matrigel的高度调控，因此不适合作为这种治疗的内部控制。因此，这些发现表明，研究人员有责任确保选择的管家基因在其特定应用中不受调节，因为这种调节可能会极大地影响其结果的准确性。本研究强调了最低限度调控内务基因（如18S rRNA）的必要性，以及竞争性模板作为RT-PCR定量应用内部控制的优越性。(C) 2001学术出版社。
Despite reports confirming cell-cycle dependent gene expression and a number of studies describing specific circumstances in which beta -actin is also regulated, the mRNA for beta -actin remains a widely used housekeeping gene internal control. Utilizing differential reverse transcriptase-polymerase chain reaction (RT-PCR), we report here the dose-dependent inhibition of beta -actin by matrigel. This was detected by comparison to the very moderate inhibition of the target gene, membrane type-1 matrix metalloproteinase (MT1-MMP), with results independently confirmed by similar findings on MT1-MMP expression using competitive RT-PCR. Furthermore, RT-PCR of the housekeeping gene 18 Svedberg Units (S) rRNA demonstrated excellent consistency, reproducibility and nonregulation by a matrigel treatment. We conclude that beta -actin is highly regulated by matrigel and therefore unsuitable as an internal control in this treatment. Hence, these findings suggest that researchers have a responsibility to ensure that the housekeeping gene of choice is not regulated in their specific application, as such regulation may dramatically affect the accuracy of their results. This study reinforces the necessity for minimally regulated housekeeping genes such as 18S rRNA, and the superiority of competitive templates as internal controls for quantitative applications of RT-PCR. (C) 2001 Academic Press.