Beta-actin--an unsuitable internal control for RT-PCR.
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 non-regulation 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.
Selvey
S
S
Genomics Research Centre, School of Health Science, Griffith University, Gold Coast, Parklands Drive, Southport, QLD 4215, Australia.
Thompson
E W
EW
Matthaei
K
K
Lea
R A
RA
Irving
M G
MG
Griffiths
L R
LR
D000199:Actins / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism* / Q000494:pharmacology; D004338:Drug Combinations; D005347:Fibroblasts; D007797:Laminin / Q000378:metabolism* / Q000494:pharmacology; D053511:Matrix Metalloproteinase 14; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D011509:Proteoglycans / Q000378:metabolism* / Q000494:pharmacology; D012337:RNA, Ribosomal, 18S / Q000235:genetics / Q000378:metabolism; D012015:Reference Standards
D000199:Actins; D004338:Drug Combinations; D007797:Laminin; C507096:Mmp14 protein, mouse; D011509:Proteoglycans; D012337:RNA, Ribosomal, 18S; C059485:matrigel; D003094:Collagen; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D053511:Matrix Metalloproteinase 14