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  • b-Actin -- an unsuitable internal control for RT-PCR

    Author(s)
    Thompson, E.
    Matthaei, K.
    Lea, Rodney
    Irving, Michael
    Griffiths, Lyn
    Griffith University Author(s)
    Irving, Michael G.
    Griffiths, Lyn
    Selvey, Saxon
    Lea, Rodney A.
    Year published
    2001
    Metadata
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    Abstract
    Despite reports confirming cell-cycle dependent gene expression and a number of studies describing specific circumstances in which ߭actin is also regulated, the mRNA for ߭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 ߭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. ...
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    Despite reports confirming cell-cycle dependent gene expression and a number of studies describing specific circumstances in which ߭actin is also regulated, the mRNA for ߭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 ߭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 ߭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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    Journal Title
    Molecular and Cellular Probes
    Volume
    15
    DOI
    https://doi.org/10.1006/mcpr.2001.0376
    Subject
    Medicinal and Biomolecular Chemistry
    Biochemistry and Cell Biology
    Publication URI
    http://hdl.handle.net/10072/3494
    Collection
    • Journal articles

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