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 β-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), β-microtubulin and S100β 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 > S100β > β-microtubulin > β-actin; by using NormFinder Excel Spreadsheet, we obtained the following sequence: GAPDH > cDNA(Oligreen) > S100β > 18S rRNA > β-actin > β-microtubulin; by using a Confidence-Interval calculation, we obtained the following sequence: cDNA(Oligreen) > 18S rRNA; GAPDH > S100β > β-microtubulin > β-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 β-actin and β-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.
Highlights
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
Reference genes and total cDNA level After induction of trauma, 10 mice were sacrificed at 30 min, 3 h, 6 h, 12 h, 24 h and 48 h, and RNA was extracted from the lesionnal zone of injured brains or from the equivalent zone of uninjuried control mice brains, reverse transcribed
The expression level of 18S rRNA, βmicrotubulin, S100β, β-actin and GAPDH were measured by real-time PCR for each individual cDNA
Summary
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. Real-time RT-PCR, which allows to measure any chosen RNA with great accuracy over a large dynamic range, has become the gold-standard for nucleic acid quantification. It has opened new investigations fields, since very small amount of RNA is needed, allowing transcripts from low-expressed genes or from very small samples to be quantified. Real-time RT-PCR gives access to the number of copies of a chosen sequence in a cDNA solution, which is obtained from RNA extracted from a known quantity of tissue. The quantification of a target gene in a given sample needs three majors steps: RNA/mRNA extraction, reverse transcription of the extracted RNA, and qPCR (quantitative PCR) processing of the synthesised cDNA. A control normalization may be performed at each step to level out dissimilarities between samples [1]
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