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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

Relative mRNA level of TRPM2 normalised to individual reference genes. TRPM2 mRNA level at (A) 5 hours and (B) 24 hours in the perihematomal brain region (RBG) of collagenase-induced ICH rats compared to saline vehicles. Bars represent mean of triplicate measurements from 5 animals per group ± SEM. Single asterisk denotes statistical significance (p < 0.05) between 24 h ICH and vehicle rats; double asterisk denotes statistical significance (p < 0.01) between 24 h ICH and vehicle rats, as assessed by t-tests.
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Figure 1: Relative mRNA level of TRPM2 normalised to individual reference genes. TRPM2 mRNA level at (A) 5 hours and (B) 24 hours in the perihematomal brain region (RBG) of collagenase-induced ICH rats compared to saline vehicles. Bars represent mean of triplicate measurements from 5 animals per group ± SEM. Single asterisk denotes statistical significance (p < 0.05) between 24 h ICH and vehicle rats; double asterisk denotes statistical significance (p < 0.01) between 24 h ICH and vehicle rats, as assessed by t-tests.

Mentions: The relative standard curve method [30] was used to calculate TRPM2 mRNA level in the perihematomal region of ICH and vehicle rats with survival times of 5 h and 24 h, relative to each of the seven reference genes individually. Figure 1a shows TRPM2 mRNA level at 5 h post-ICH. Large variations were observed depending on which reference gene was used for normalisation. At the 24 hour time point, when TRPM2 data were normalised to GUSB only, a significant (p < 0.01) 1.85-fold increase in mean TRPM2 mRNA level was observed in the collagenase ICH rats compared to saline vehicles (Figure 1b). A significant (p < 0.05) 1.4-fold increase was found in TRPM2 mRNA level when data were normalised to HPRT only. When each of the other reference genes was used individually for normalisation, there were no significant differences. Given the discrepancy in these results and the large variation between samples, we proceeded with a reference gene evaluation study to determine the most stable reference genes in the collagenase model of ICH.


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)

Relative mRNA level of TRPM2 normalised to individual reference genes. TRPM2 mRNA level at (A) 5 hours and (B) 24 hours in the perihematomal brain region (RBG) of collagenase-induced ICH rats compared to saline vehicles. Bars represent mean of triplicate measurements from 5 animals per group ± SEM. Single asterisk denotes statistical significance (p < 0.05) between 24 h ICH and vehicle rats; double asterisk denotes statistical significance (p < 0.01) between 24 h ICH and vehicle rats, as assessed by t-tests.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Relative mRNA level of TRPM2 normalised to individual reference genes. TRPM2 mRNA level at (A) 5 hours and (B) 24 hours in the perihematomal brain region (RBG) of collagenase-induced ICH rats compared to saline vehicles. Bars represent mean of triplicate measurements from 5 animals per group ± SEM. Single asterisk denotes statistical significance (p < 0.05) between 24 h ICH and vehicle rats; double asterisk denotes statistical significance (p < 0.01) between 24 h ICH and vehicle rats, as assessed by t-tests.
Mentions: The relative standard curve method [30] was used to calculate TRPM2 mRNA level in the perihematomal region of ICH and vehicle rats with survival times of 5 h and 24 h, relative to each of the seven reference genes individually. Figure 1a shows TRPM2 mRNA level at 5 h post-ICH. Large variations were observed depending on which reference gene was used for normalisation. At the 24 hour time point, when TRPM2 data were normalised to GUSB only, a significant (p < 0.01) 1.85-fold increase in mean TRPM2 mRNA level was observed in the collagenase ICH rats compared to saline vehicles (Figure 1b). A significant (p < 0.05) 1.4-fold increase was found in TRPM2 mRNA level when data were normalised to HPRT only. When each of the other reference genes was used individually for normalisation, there were no significant differences. Given the discrepancy in these results and the large variation between samples, we proceeded with a reference gene evaluation study to determine the most stable reference genes in the collagenase model of ICH.

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