β-actin -: an unsuitable internal control for RT-PCR

被引:322
作者
Selvey, S
Thompson, EW
Matthaei, K
Lea, RA
Irving, MG
Griffiths, LR
机构
[1] Griffith Univ, Genom Res Ctr, Gold Coast, Qld 4217, Australia
[2] Univ Melbourne, St Vincents Inst Med Res, VBCRC Invas & Metastasis Unit, Melbourne, Vic 3000, Australia
[3] Australian Natl Univ, John Curtin Sch Med Res, Canberra, ACT 2600, Australia
关键词
beta-actin; RT-PCR; housekeeping gene; quantitation;
D O I
10.1006/mcpr.2001.0376
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:307 / 311
页数:5
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