β-Actin—an unsuitable internal control for RT-PCR
Selvey, S., Thompson, Erik W., Matthaei, K., Lea, Rod A., Irving, M. G., & Griffiths, Lyn R. (2001) β-Actin—an unsuitable internal control for RT-PCR. Molecular and Cellular Probes, 15(5), pp. 307-311.
Despite reports confirming cell-cycle dependent gene expression and a number of studies describing specific circumstances in which β-actin is also regulated, the mRNA for β-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 β-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 non-regulation by a matrigel treatment. We conclude that β-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.
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|Item Type:||Journal Article|
|Divisions:||Current > Schools > School of Biomedical Sciences
Current > QUT Faculties and Divisions > Faculty of Health
Current > Institutes > Institute of Health and Biomedical Innovation
|Copyright Owner:||Copyright 2001 Academic Press (Elsevier)|
|Deposited On:||24 Sep 2013 01:51|
|Last Modified:||12 Jun 2014 01:25|
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