Selection of Endogenous Control Genes for Normalization of Gene Expression Analysis after Experimental Brain Trauma in Mice

Abstract

Quantitative measurements of gene expression require correction for tissue sample size, RNA quantity, and reverse transcription efficiency. This can be achieved by normalization with control genes. The study was designed to identify candidates not altered after brain trauma. Male C57Bl/6 mice were anesthetized with isoflurane, and a pneumatic brain trauma was induced by controlled cortical impact (CCI) on the right parietal cortex. Brains were removed at 15 min, and 3, 6, 12 and 24 h after CCI and from naive animals (n = 6 each). Absolute copies of six control genes (beta-2-microglobin [B2M], cyclophilin A, beta-actin, hypoxanthine ribosyltransferase [HPRT], porphobilinogen deaminase [PBGD], and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and one example target gene (iNOS) were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR; Lightcycler) in the traumatic focus and contralateral tissue. Control gene expression was stable until 12 h after CCI. At 24 h after CCI expression of B2M, cyclophilin A and HPRT remained stable in the contusion, while expression of beta-actin, GAPDH, and PBGD increased. Due to variations between animals (+/-85%), increases in beta-actin (+64%) and GAPDH (+59%) did not reach the level of significance. In non-contused tissue, expression of all genes dropped 24 h after CCI (range, -17% to -61%). Due to low variations between animals and stable expression after CCI, B2M and cyclophilin A seem to be suitable to serve as single normalizer. Normalization of the example target gene iNOS resulted in varying relative expression extending from onefold (PBDG) to 10-fold (HPRT). The results suggest that the knowledge of the temporal profile of control genes is essential to properly interpret results of mRNA quantification.

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

“…However, the potential for variable expression of b-actin in sham-control cases suggests that it is not a good reference gene for FPI studies examining relative change in certain transcripts. In contrast to the mouse CCI model (Thal et al, 2008), our results show rat cyclophilin A as a poor reference gene for diffuse TBI. Notably, it was the best of those tested for use with the UEC targeted deafferentation model.…”

“…An ideal reference gene should be stably expressed across all tissues, developmental stages, and experimental conditions, a requirement that is difficult to confirm in real practice. Prior studies have shown that CNS expression of some of these widely used reference genes may be altered in models of hypoxia=ischemia (Zhong and Simons, 1999;Bond et al, 2002;Kobayashi et al, 2004), seizure (Chen et al, 2001), hippocampal deafferentation (Phillips et al, 1987;Phillips and Steward, 1990), demyelination (Meldgaard et al, 2006), and TBI (Thal et al, 2008;Rhinn et al, 2008;Cook et al, 2008). To make the choice more difficult, other brain trauma paradigms report some of these reference genes to be invariant and useful for qRT-PCR normalization.…”

“…This approach requires determination of reference efficacy for each candidate gene, and while it can help to identify optimal choices for RNA normalization, the examination of a targeted gene of interest in this context may result in reduced detection of experimental effect, particularly if the change in transcript is small. Two additional studies have examined qRT-PCR normalization after TBI (Thal et al, 2008;Rhinn et al, 2008); however, these have also been limited to acute 1-to 3-days-post-injury intervals, and focused on single rodent models. TBI presents a particular challenge for the accurate quantification of mRNA due to the fact that heterogeneous cell populations are injured, generating a multifaceted pathology (primary tissue damage, progressive secondary degeneration, ischemic=hypoxic damage, edema, hemorrhage, and inflammation).…”

“…Several prior studies of gene expression during injuryinduced synaptic plasticity (Kerber et al, 2003;Price et al, 2003;Molteni et al, 2002;Blain et al, 2004) employ qRT-PCR reference genes recently reported to vary with brain trauma (Thal et al, 2008;Rhinn et al, 2008;Cook et al, 2008). In the present study we have extended TBI reference gene analysis, now focusing on 2-15 days post-injury, the period of functional synaptic recovery.…”

Developmental expression of P5 ATPase mRNA in the mouse

“…HPRT expression reflects the average expression of multiple common reference genes, making it the best choice when using only a single normalization gene [22]. HPRT has also been shown to be expressed at constant levels compared to other reference genes [21,[23][24][25]. To determine the stability of HPRT in all developmental stages used, its expression was validated against additional commonly used reference genes, including B2m (beta-2-microglobulin), Gapdh (glyceraldehyde 3-phosphate dehydrogenase), Gusb (beta-glucuronidase), Tfrc (transferrin receptor), and Pgk1 (phosphoglycerate kinase 1) [21,26].…”