ArticleBMC molecular biology2008
Housekeeping while brain's storming Validation of normalizing factors for gene expression studies in a murine model of traumatic brain injury.
Article in BMC molecular biology, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 38 papers.
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38 citing papers in PubMed, 67 citations in OpenAlex.
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- Systematic Identification of the Optimal Housekeeping Genes for Accurate Transcriptomic and Proteomic Profiling of Tissues following Complex Traumatic Injury.Methods and protocols · 2023Article
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- Loop extrusion promotes an alternate pathway for isotype switching.Cell reports · 2021Article
- Effects of fibrinogen synthesis inhibition on vascular cognitive impairment during traumatic brain injury in mice.Brain research · 2021Article
- Validation of reference genes for expression analysis in a murine trauma model combining traumatic brain injury and femoral fracture.Scientific reports · 2020Article
- Selection of Reliable Reference Genes for Analysis of Gene Expression in Spinal Cord during Rat Postnatal Development and after Injury.Brain sciences · 2019Article
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- MBD4 Facilitates Immunoglobulin Class Switch Recombination.Molecular and cellular biology · 2017Article
- β-endorphin regulates alcohol consumption induced by exercise restriction in female mice.Alcohol (Fayetteville, N.Y.) · 2016Article
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- Reference genes for reverse transcription quantitative PCR in canine brain tissue.BMC research notes · 2015Article
- Constraints contributed by chromatin looping limit recombination targeting during Ig class switch recombination.Journal of immunology (Baltimore, Md. : 1950) · 2015Article
- Altering the redox state of skeletal muscle by glutathione depletion increases the exercise-activation of PGC-1α.Physiological reports · 2014Article
- Alpha II Spectrin breakdown products in immature Sprague Dawley rat hippocampus and cortex after traumatic brain injury.Brain research · 2014Article
- Maternal nutritional history modulates the hepatic IGF-IGFBP axis in adult male rat offspring.Endocrine · 2014Article
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Authors and funding
6 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundTraumatic 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.
resultsWe 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.
conclusionThis 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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