beta-actin - an unsuitable internal control for RT-PCR

Despite reports confirming cell-cycle dependent gene expression and a numbe r of studies describing specific circumstances in which beta -actin is also regulated, the mRNA for beta -actin remains a widely used housekeeping gen e internal control. Utilizing differential reverse transcriptase-polymerase chain reaction (RT-PCR), we report here the dose-dependent inhibition of b eta -actin by matrigel. This was detected by comparison to the very moderat e inhibition of the target gene, membrane type-1 matrix metalloproteinase ( MT1-MMP), with results independently confirmed by similar findings on MT1-M MP expression using competitive RT-PCR. Furthermore, RT-PCR of the housekee ping gene 18 Svedberg Units (S) rRNA demonstrated excellent consistency, re producibility and nonregulation by a matrigel treatment. We conclude that b eta -actin is highly regulated by matrigel and therefore unsuitable as an i nternal control in this treatment. Hence, these findings suggest that resea rchers have a responsibility to ensure that the housekeeping gene of choice is not regulated in their specific application, as such regulation may dra matically affect the accuracy of their results. This study reinforces the n ecessity for minimally regulated housekeeping genes such as 18S rRNA, and t he superiority of competitive templates as internal controls for quantitati ve applications of RT-PCR. (C) 2001 Academic Press.