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Reference genes for normalising gene expression data in collagenase-induced rat intracerebral haemorrhage.

Cook NL, Kleinig TJ, van den Heuvel C, Vink R - BMC Mol. Biol. (2010)

Bottom Line: Reference gene panels have therefore been proposed to overcome this potential confounder.When normalised to the four most stable reference genes selected for accurate normalisation of data, we found no significant difference between ICH and vehicle rats.The panel of reference genes identified in the present study will enable more accurate normalisation of gene expression data in the acute phase of experimental ICH.

View Article: PubMed Central - HTML - PubMed

Affiliation: Discipline of Anatomy and Pathology, School of Medical Sciences, The University of Adelaide, Adelaide, SA 5005, Australia.

ABSTRACT

Background: The mechanisms of brain injury following intracerebral haemorrhage (ICH) are incompletely understood. Gene expression studies using quantitative real-time RT-PCR following ICH have increased our understanding of these mechanisms, however the inconsistent results observed may be related to inappropriate reference gene selection. Reference genes should be stably expressed across different experimental conditions, however, transcript levels of common reference genes have been shown to vary considerably. Reference gene panels have therefore been proposed to overcome this potential confounder.

Results: The present study evaluated the stability of seven candidate reference genes in the striatum and overlying cortex of collagenase-induced ICH in rodents at survival times of 5 and 24 hours. Transcript levels of the candidate reference genes were quantified and ranked in order of stability using geNorm. When our gene of interest, transient receptor potential melastatin 2 (TRPM2), was normalised against each reference gene individually, TRPM2 mRNA levels were highly variable. When normalised to the four most stable reference genes selected for accurate normalisation of data, we found no significant difference between ICH and vehicle rats.

Conclusion: The panel of reference genes identified in the present study will enable more accurate normalisation of gene expression data in the acute phase of experimental ICH.

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Related in: MedlinePlus

Average real-time RT-PCR cycle threshold (Ct) values of candidate reference genes. Data are displayed as average raw Ct (not yet normalised) values from individual groups of collagenase ICH and saline vehicle rats. The line in the box indicates the median, while the box represents the 25th and 75th percentile. Whiskers represent the maximum and minimum values.
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Figure 3: Average real-time RT-PCR cycle threshold (Ct) values of candidate reference genes. Data are displayed as average raw Ct (not yet normalised) values from individual groups of collagenase ICH and saline vehicle rats. The line in the box indicates the median, while the box represents the 25th and 75th percentile. Whiskers represent the maximum and minimum values.

Mentions: Figure 3 shows the average raw (i.e. not yet normalised) Ct values of the collagenase ICH and saline vehicle rats for each candidate reference gene. The raw Ct values of GAPDH were fairly consistent between groups, while HPRT exhibited more variation. The collagenase ICH samples generally had more raw Ct variation than the saline vehicle samples. In Figure 4, the raw Ct values were normalised to the geometric mean of the four most stable reference genes determined by geNorm (B2MG, GUSB, POL2R and GAPDH), in order to obtain the relative mRNA level of each reference gene. One-way ANOVA revealed statistically significant differences in relative mRNA level within the SDHA group.


Reference genes for normalising gene expression data in collagenase-induced rat intracerebral haemorrhage.

Cook NL, Kleinig TJ, van den Heuvel C, Vink R - BMC Mol. Biol. (2010)

Average real-time RT-PCR cycle threshold (Ct) values of candidate reference genes. Data are displayed as average raw Ct (not yet normalised) values from individual groups of collagenase ICH and saline vehicle rats. The line in the box indicates the median, while the box represents the 25th and 75th percentile. Whiskers represent the maximum and minimum values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2823748&req=5

Figure 3: Average real-time RT-PCR cycle threshold (Ct) values of candidate reference genes. Data are displayed as average raw Ct (not yet normalised) values from individual groups of collagenase ICH and saline vehicle rats. The line in the box indicates the median, while the box represents the 25th and 75th percentile. Whiskers represent the maximum and minimum values.
Mentions: Figure 3 shows the average raw (i.e. not yet normalised) Ct values of the collagenase ICH and saline vehicle rats for each candidate reference gene. The raw Ct values of GAPDH were fairly consistent between groups, while HPRT exhibited more variation. The collagenase ICH samples generally had more raw Ct variation than the saline vehicle samples. In Figure 4, the raw Ct values were normalised to the geometric mean of the four most stable reference genes determined by geNorm (B2MG, GUSB, POL2R and GAPDH), in order to obtain the relative mRNA level of each reference gene. One-way ANOVA revealed statistically significant differences in relative mRNA level within the SDHA group.

Bottom Line: Reference gene panels have therefore been proposed to overcome this potential confounder.When normalised to the four most stable reference genes selected for accurate normalisation of data, we found no significant difference between ICH and vehicle rats.The panel of reference genes identified in the present study will enable more accurate normalisation of gene expression data in the acute phase of experimental ICH.

View Article: PubMed Central - HTML - PubMed

Affiliation: Discipline of Anatomy and Pathology, School of Medical Sciences, The University of Adelaide, Adelaide, SA 5005, Australia.

ABSTRACT

Background: The mechanisms of brain injury following intracerebral haemorrhage (ICH) are incompletely understood. Gene expression studies using quantitative real-time RT-PCR following ICH have increased our understanding of these mechanisms, however the inconsistent results observed may be related to inappropriate reference gene selection. Reference genes should be stably expressed across different experimental conditions, however, transcript levels of common reference genes have been shown to vary considerably. Reference gene panels have therefore been proposed to overcome this potential confounder.

Results: The present study evaluated the stability of seven candidate reference genes in the striatum and overlying cortex of collagenase-induced ICH in rodents at survival times of 5 and 24 hours. Transcript levels of the candidate reference genes were quantified and ranked in order of stability using geNorm. When our gene of interest, transient receptor potential melastatin 2 (TRPM2), was normalised against each reference gene individually, TRPM2 mRNA levels were highly variable. When normalised to the four most stable reference genes selected for accurate normalisation of data, we found no significant difference between ICH and vehicle rats.

Conclusion: The panel of reference genes identified in the present study will enable more accurate normalisation of gene expression data in the acute phase of experimental ICH.

Show MeSH
Related in: MedlinePlus