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Housekeeping while brain's storming Validation of normalizing factors for gene expression studies in a murine model of traumatic brain injury.

Rhinn H, Marchand-Leroux C, Croci N, Plotkine M, Scherman D, Escriou V - BMC Mol. Biol. (2008)

Bottom Line: We have compared five potential reference genes as well as total cDNA level monitored using Oligreen reagent in order to determine the best normalizing factors for quantitative RT-PCR expression studies in the early phase (0-48 h post-trauma (PT)) of a murine model of diffuse brain injury.The potential of total cDNA as measured by Oligreen as a first-intention normalizing factor with a broad field of applications is highlighted.A generic time- and cost-effective procedure for normalization factor validation is proposed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Inserm, U640, Paris, F-75006 France. herve.rhinn@gmail.com

ABSTRACT

Background: Traumatic brain injury models are widely studied, especially through gene expression, either to further understand implied biological mechanisms or to assess the efficiency of potential therapies. A large number of biological pathways are affected in brain trauma models, whose elucidation might greatly benefit from transcriptomic studies. However the suitability of reference genes needed for quantitative RT-PCR experiments is missing for these models.

Results: We have compared five potential reference genes as well as total cDNA level monitored using Oligreen reagent in order to determine the best normalizing factors for quantitative RT-PCR expression studies in the early phase (0-48 h post-trauma (PT)) of a murine model of diffuse brain injury. The levels of 18S rRNA, and of transcripts of beta-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), beta-microtubulin and S100beta were determined in the injured brain region of traumatized mice sacrificed at 30 min, 3 h, 6 h, 12 h, 24 h and 48 h post-trauma. The stability of the reference genes candidates and of total cDNA was evaluated by three different methods, leading to the following rankings as normalization factors, from the most suitable to the less: by using geNorm VBA applet, we obtained the following sequence: cDNA(Oligreen); GAPDH > 18S rRNA > S100beta > beta-microtubulin > beta-actin; by using NormFinder Excel Spreadsheet, we obtained the following sequence: GAPDH > cDNA(Oligreen) > S100beta > 18S rRNA > beta-actin > beta-microtubulin; by using a Confidence-Interval calculation, we obtained the following sequence: cDNA(Oligreen) > 18S rRNA; GAPDH > S100beta > beta-microtubulin > beta-actin.

Conclusion: This work suggests that Oligreen cDNA measurements, 18S rRNA and GAPDH or a combination of them may be used to efficiently normalize qRT-PCR gene expression in mouse brain trauma injury, and that beta-actin and beta-microtubulin should be avoided. The potential of total cDNA as measured by Oligreen as a first-intention normalizing factor with a broad field of applications is highlighted. Pros and cons of the three methods of normalization factors selection are discussed. A generic time- and cost-effective procedure for normalization factor validation is proposed.

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Chosen primers.
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Figure 7: Chosen primers.

Mentions: PCR primers for tested reference gene were chosen in published articles, for their common use as reference genes and their belonging to different biological pathways (Fig. 7). Real-time PCR reactions were carried out using ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) in a 384-well, clear optical reaction plate with optical adhesive covers (Applied Biosystems). Reactions were run in a 5 μl volume in duplicate, with 2 μL of cDNA solution and 3 μL of a homemade target-specific mix composed of 5/6 2× Power SYBR Green Master Mix (Applied Biosystems) and 1/6 of 100 mM primers solution. The PCR program was: 95°C for 10 min, followed by 45 cycles of (15 seconds at 95°C; 1 min at 60°C). Product specificity was assessed by 3% agarose gel electrophoresis followed by ethidium bromide staining.


Housekeeping while brain's storming Validation of normalizing factors for gene expression studies in a murine model of traumatic brain injury.

Rhinn H, Marchand-Leroux C, Croci N, Plotkine M, Scherman D, Escriou V - BMC Mol. Biol. (2008)

Chosen primers.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2500043&req=5

Figure 7: Chosen primers.
Mentions: PCR primers for tested reference gene were chosen in published articles, for their common use as reference genes and their belonging to different biological pathways (Fig. 7). Real-time PCR reactions were carried out using ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) in a 384-well, clear optical reaction plate with optical adhesive covers (Applied Biosystems). Reactions were run in a 5 μl volume in duplicate, with 2 μL of cDNA solution and 3 μL of a homemade target-specific mix composed of 5/6 2× Power SYBR Green Master Mix (Applied Biosystems) and 1/6 of 100 mM primers solution. The PCR program was: 95°C for 10 min, followed by 45 cycles of (15 seconds at 95°C; 1 min at 60°C). Product specificity was assessed by 3% agarose gel electrophoresis followed by ethidium bromide staining.

Bottom Line: We have compared five potential reference genes as well as total cDNA level monitored using Oligreen reagent in order to determine the best normalizing factors for quantitative RT-PCR expression studies in the early phase (0-48 h post-trauma (PT)) of a murine model of diffuse brain injury.The potential of total cDNA as measured by Oligreen as a first-intention normalizing factor with a broad field of applications is highlighted.A generic time- and cost-effective procedure for normalization factor validation is proposed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Inserm, U640, Paris, F-75006 France. herve.rhinn@gmail.com

ABSTRACT

Background: Traumatic brain injury models are widely studied, especially through gene expression, either to further understand implied biological mechanisms or to assess the efficiency of potential therapies. A large number of biological pathways are affected in brain trauma models, whose elucidation might greatly benefit from transcriptomic studies. However the suitability of reference genes needed for quantitative RT-PCR experiments is missing for these models.

Results: We have compared five potential reference genes as well as total cDNA level monitored using Oligreen reagent in order to determine the best normalizing factors for quantitative RT-PCR expression studies in the early phase (0-48 h post-trauma (PT)) of a murine model of diffuse brain injury. The levels of 18S rRNA, and of transcripts of beta-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), beta-microtubulin and S100beta were determined in the injured brain region of traumatized mice sacrificed at 30 min, 3 h, 6 h, 12 h, 24 h and 48 h post-trauma. The stability of the reference genes candidates and of total cDNA was evaluated by three different methods, leading to the following rankings as normalization factors, from the most suitable to the less: by using geNorm VBA applet, we obtained the following sequence: cDNA(Oligreen); GAPDH > 18S rRNA > S100beta > beta-microtubulin > beta-actin; by using NormFinder Excel Spreadsheet, we obtained the following sequence: GAPDH > cDNA(Oligreen) > S100beta > 18S rRNA > beta-actin > beta-microtubulin; by using a Confidence-Interval calculation, we obtained the following sequence: cDNA(Oligreen) > 18S rRNA; GAPDH > S100beta > beta-microtubulin > beta-actin.

Conclusion: This work suggests that Oligreen cDNA measurements, 18S rRNA and GAPDH or a combination of them may be used to efficiently normalize qRT-PCR gene expression in mouse brain trauma injury, and that beta-actin and beta-microtubulin should be avoided. The potential of total cDNA as measured by Oligreen as a first-intention normalizing factor with a broad field of applications is highlighted. Pros and cons of the three methods of normalization factors selection are discussed. A generic time- and cost-effective procedure for normalization factor validation is proposed.

Show MeSH
Related in: MedlinePlus