Appropriate 'housekeeping' genes for use in expression profiling the effects of environmental estrogens in fish
Identifieur interne : 000E89 ( Main/Exploration ); précédent : 000E88; suivant : 000E90Appropriate 'housekeeping' genes for use in expression profiling the effects of environmental estrogens in fish
Auteurs : Amy L. Filby [Royaume-Uni] ; Charles R. Tyler [Royaume-Uni]Source :
- BMC Molecular Biology [ 1471-2199 ] ; 2007.
Abstract
Attempts to develop a mechanistic understanding of the effects of environmental estrogens on fish are increasingly conducted at the level of gene expression. Appropriate application of real-time PCR in such studies requires the use of a stably expressed 'housekeeping' gene as an internal control to normalize for differences in the amount of starting template between samples.
We sought to identify appropriate genes for use as internal controls in experimental treatments with estrogen by analyzing the expression of eight functionally distinct 'housekeeping' genes (18S ribosomal RNA [18S rRNA],
Based on the data presented, we recommend 18S rRNA,
Url:
DOI: 10.1186/1471-2199-8-10
PubMed: 17288598
PubMed Central: 1802086
Affiliations:
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Le document en format XML
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<front><div type="abstract" xml:lang="en"><sec><title>Background</title>
<p>Attempts to develop a mechanistic understanding of the effects of environmental estrogens on fish are increasingly conducted at the level of gene expression. Appropriate application of real-time PCR in such studies requires the use of a stably expressed 'housekeeping' gene as an internal control to normalize for differences in the amount of starting template between samples.</p>
</sec>
<sec><title>Results</title>
<p>We sought to identify appropriate genes for use as internal controls in experimental treatments with estrogen by analyzing the expression of eight functionally distinct 'housekeeping' genes (18S ribosomal RNA [18S rRNA], <italic>ribosomal protein l8 </italic>
[<italic>rpl8</italic>
], <italic>elongation factor 1 alpha </italic>
[<italic>ef1a</italic>
], <italic>glucose-6-phosphate dehydrogenase </italic>
[<italic>g6pd</italic>
], <italic>beta actin </italic>
[<italic>bactin</italic>
], <italic>glyceraldehyde-3-phosphate dehydrogenase </italic>
[<italic>gapdh</italic>
], <italic>hypoxanthine phosphoribosyltransferase 1 </italic>
[<italic>hprt1</italic>
], and <italic>tata box binding protein </italic>
[<italic>tbp</italic>
]) following exposure to the environmental estrogen, 17α-ethinylestradiol (EE<sub>2</sub>
), in the fathead minnow (<italic>Pimephales promelas</italic>
). Exposure to 10 ng/L EE<sub>2 </sub>
for 21 days down-regulated the expression of <italic>ef1a</italic>
, <italic>g6pd</italic>
, <italic>bactin </italic>
and <italic>gapdh </italic>
in the liver, and <italic>bactin </italic>
and <italic>gapdh </italic>
in the gonad. Some of these effects were gender-specific, with <italic>bactin </italic>
in the liver and <italic>gapdh </italic>
in the gonad down-regulated by EE<sub>2 </sub>
in males only. Furthermore, when <italic>ef1a</italic>
, <italic>g6pd</italic>
, <italic>bactin </italic>
or <italic>gapdh </italic>
were used for normalization, the hepatic expression of two genes of interest, <italic>vitellogenin </italic>
(<italic>vtg</italic>
) and <italic>cytochrome P450 1A </italic>
(<italic>cyp1a</italic>
) following exposure to EE<sub>2 </sub>
was overestimated.</p>
</sec>
<sec><title>Conclusion</title>
<p>Based on the data presented, we recommend 18S rRNA, <italic>rpl8</italic>
, <italic>hprt1 </italic>
and/or <italic>tbp</italic>
, but not <italic>ef1a</italic>
, <italic>g6pd</italic>
, <italic>bactin </italic>
and/or <italic>gapdh</italic>
, as likely appropriate internal controls in real-time PCR studies of estrogens effects in fish. Our studies show that pre-validation of control genes considering the scope and nature of the experiments to be performed, including both gender and tissue type, is critical for accurate assessments of the effects of environmental estrogens on gene expression in fish.</p>
</sec>
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