Injury Modality, Survival Interval and Sample Region are Critical Determinants of qRT-PCR Reference Gene Selection during Long-Term Recovery from Brain Trauma

In the present study we examined expression of four real-time quantitative RT-PCR reference genes commonly applied to rodent models of brain injury. Transcripts for β-actin, cyclophilin A, GAPDH, and 18S rRNA were assessed at 2-15 d postinjury, focusing on the period of synaptic recovery. Diffuse moderate central fluid percussion injury (FPI) was contrasted with unilateral entorhinal cortex lesion (UEC), a model of targeted deafferentation. Expression in UEC hippocampus, as well as in FPI hippocampus and parietotemporal cortex was analyzed by qRT-PCR. Within-group variability of gene expression was assessed and change in expression relative to paired controls determined. None of the four common reference genes tested was invariant across brain region, survival time and type of injury. Cyclophilin A appeared appropriate as a reference gene in UEC hippocampus, while β-actin was most stable for the hippocampus subjected to FPI. However, each gene may fail as a suitable reference with certain test genes whose RNA expression is targeted for measurement. In FPI cortex, all reference genes were significantly altered over time, compromising their utility for time course studies. Despite such temporal variability, certain genes may be appropriate references if limited to single survival times. These data provide an extended baseline for identification of appropriate reference genes in rodent studies of recovery from brain injury. In this context, we outline additional considerations for selecting a qRT-PCR normalization strategy in such studies. As previously concluded for acute postinjury intervals, we stress the importance of reference gene validation for each brain injury paradigm and each set of experimental conditions. Keywords: traumatic brain injury, genomics, molecular biological approaches, neuroplasticity, synaptic loss and deafferentation