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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

Normalised mRNA level of TRPM2 relative to multiple reference genes. TRPM2 mRNA level in the perihematomal brain region (RBG) of collagenase-induced ICH animals was compared to saline vehicle controls, at 5 h and 24 h survival times. The most stable reference genes determined by geNorm were used for normalisation in each group (5 h: GAPDH, HPRT, POL2R, SDHA; 24 h: B2MG, GUSB, POL2R). Bars represent mean of triplicate measurements from 5 animals, ± SEM.
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Figure 5: Normalised mRNA level of TRPM2 relative to multiple reference genes. TRPM2 mRNA level in the perihematomal brain region (RBG) of collagenase-induced ICH animals was compared to saline vehicle controls, at 5 h and 24 h survival times. The most stable reference genes determined by geNorm were used for normalisation in each group (5 h: GAPDH, HPRT, POL2R, SDHA; 24 h: B2MG, GUSB, POL2R). Bars represent mean of triplicate measurements from 5 animals, ± SEM.

Mentions: The qBasePlus program was used to calculate the normalised mRNA level of TRPM2 in the perihematomal brain region at 5 h and 24 h post-ICH, relative to the most stable reference genes for each time point as determined by geNorm (5 h: GAPDH, HPRT, POL2R and SDHA; 24 h: B2MG, GUSB and POL2R). qBasePlus utilises a modified version of the 2-ΔΔCt method of relative expression analysis [37] that takes into account multiple reference genes and gene-specific amplification efficiencies [38]. There was no significant difference in mean TRPM2 transcript levels between collagenase ICH and saline vehicle animals (Figure 5).


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)

Normalised mRNA level of TRPM2 relative to multiple reference genes. TRPM2 mRNA level in the perihematomal brain region (RBG) of collagenase-induced ICH animals was compared to saline vehicle controls, at 5 h and 24 h survival times. The most stable reference genes determined by geNorm were used for normalisation in each group (5 h: GAPDH, HPRT, POL2R, SDHA; 24 h: B2MG, GUSB, POL2R). Bars represent mean of triplicate measurements from 5 animals, ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Normalised mRNA level of TRPM2 relative to multiple reference genes. TRPM2 mRNA level in the perihematomal brain region (RBG) of collagenase-induced ICH animals was compared to saline vehicle controls, at 5 h and 24 h survival times. The most stable reference genes determined by geNorm were used for normalisation in each group (5 h: GAPDH, HPRT, POL2R, SDHA; 24 h: B2MG, GUSB, POL2R). Bars represent mean of triplicate measurements from 5 animals, ± SEM.
Mentions: The qBasePlus program was used to calculate the normalised mRNA level of TRPM2 in the perihematomal brain region at 5 h and 24 h post-ICH, relative to the most stable reference genes for each time point as determined by geNorm (5 h: GAPDH, HPRT, POL2R and SDHA; 24 h: B2MG, GUSB and POL2R). qBasePlus utilises a modified version of the 2-ΔΔCt method of relative expression analysis [37] that takes into account multiple reference genes and gene-specific amplification efficiencies [38]. There was no significant difference in mean TRPM2 transcript levels between collagenase ICH and saline vehicle animals (Figure 5).

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