The quantification of gene expression in an animal model of brain ischaemia using TaqMan™ real-time RT-PCR

Bond, Brian; Virley, David; Cairns, Nigel J.; Hunter, A. Jackie; Moore, Gary B.T.; Moss, Stephen J.; Mudge, Anne W.; Walsh, Frank S.; Jazin, Elena; Preece, Paul

doi:10.1016/s0169-328x(02)00417-5

Cited by 78 publications

(58 citation statements)

References 38 publications

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“…For genes whose mRNA was reported to be unaltered or decreased, it would be impossible to say whether they were actually altered or not. Changes in housekeeping genes are reported in other diseases as well and the present results suggest that relevant standards should be determined specifically in each disease and model (Bond et al, 2002).…”

Section: Discussionsupporting

confidence: 59%

Induction of housekeeping gene expression after subarachnoid hemorrhage in dogs

Aihara

Jahromi

Yassari

et al. 2008

Journal of Neuroscience Methods

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Changes in gene expression are commonly assessed relative to the expression of housekeeping genes, which are assumed to remain unchanged. We tested this assumption in cerebral arteries obtained from dogs 4 and 7 days after SAH had been created using the double hemorrhage model. Basilar arteries were removed and examined for expression of messenger ribonucleic acid (mRNA) levels using quantitative real-time polymerase chain reaction. Cross sections of basilar arteries were stained immunohistochemically for proliferating cell nuclear antigen (PCNA) and 4′,6-diamidino-2-phenylindole (DAPI). Positively-stained cells were counted and numbers obtained were normalized to the cross-sectional area. Results were compared to normal dog basilar arteries contracted pharmacologically in vitro. SAH resulted in significant vasospasm (P<0.001 for each, paired t-tests). There were significant increases in mRNA for β-actin (441%, P=0.01), glyceraldehyde-3-phosphate dehydrogenase (566%, P=0.007) and 18S ribosomal RNA (320%, P=0.025) 7 days after SAH. Total mRNA was increased 7 days after SAH relative to genomic DNA (157%, P=0.009). There were significant increases in the number of cells in the tunica media and adventitia of arteries after SAH and a significant decrease in the media after contraction in vitro. Cells in the tunica media and adventitia labeled with PCNA were significantly increased at both times after SAH. Transcripts for housekeeping genes are increased after SAH, making standardization to them potentially invalid. The increase is due to proliferation of cells in the adventitia and increased total mRNA in the media and adventitia.

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Section: Discussionsupporting

confidence: 59%

Induction of housekeeping gene expression after subarachnoid hemorrhage in dogs

Aihara

Jahromi

Yassari

et al. 2008

Journal of Neuroscience Methods

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“…A majority of TBI qRT-PCR studies use one of these reference genes for quantitative evaluation of gene expression (Rhinn et al, 2008). Expression of such genes in target tissue is assumed unchanged by experimental treatment, and differences in these genes serve to control for variance in sample quality (Bond et al, 2002;Wilson, 1997). Using high-quality RNA and a consistent RT-PCR protocol, we found that each reference gene tested was specifically altered by focal deafferentation or diffuse brain injury.…”

Section: Discussionmentioning

confidence: 99%

“…In the context of long-term TBI recovery, this means no difference in injured:control ratio for all time points included, no overall change during the postinjury time course selected, and no change in reference gene as a function of experimental treatment condition (e.g., therapeutic drug effect). Different types of reference gene alteration can profoundly affect outcome (Bond et al, 2002). For example, if the reference and target gene are similarly shifted (i.e., both increased or both decreased), normalization can produce a falsenegative result.…”

Section: Validation Of Reference Genes For Long-term Recovery Studiesmentioning

confidence: 99%

“…For example, after careful determination of our input RNA quality and quantity, we show that significant shifts of 12-20% in phosphacan and sRPTP-b following UEC lesion can be detected by this method of normalization. Depending on the overall data distribution, small increases or decreases in a gene may be no less biologically significant than large quantitative changes (Bond et al, 2002). Acceptable variability cannot be defined a priori, but must be dictated by the resolution required to detect injury effect.…”

Section: Validation Of Reference Genes For Long-term Recovery Studiesmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Harris

Reeves

Phillips

2009

Journal of Neurotrauma

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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 b-actin, cyclophilin A, GAPDH, and 18S rRNA were assessed at 2-15 days post-injury, 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 was 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 b-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 post-injury intervals, we stress the importance of reference gene validation for each brain injury paradigm and each set of experimental conditions.

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Transcription and pathway analysis of the superior temporal cortex and anterior prefrontal cortex in schizophrenia

Barnes

Huxley-Jones

Maycox

et al. 2011

J of Neuroscience Research

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The molecular basis of schizophrenia is poorly understood; however, different brain regions are believed to play distinct roles in disease symptomology. We have studied gene expression in the superior temporal cortex (Brodmann area 22; BA22), which may play a role in positive pathophysiology, and compared our results with data from the anterior prefrontal cortex (BA10), which shows evidence for a role in negative symptoms. Genome-wide mRNA expression was determined in the BA22 region in 23 schizophrenics and 19 controls and compared with a BA10 data set from the same subjects. After adjustments for confounding sources of variation, we carried out GeneGO pathway enrichment analysis in each region. Significant differences were seen in age-related transcriptional changes between the BA22 and the BA10 regions, 21.8% and 41.4% of disease-associated transcripts showing age association, respectively. After removing age associated changes from our data, we saw the highest enrichment in processes mediating cell adhesion, synaptic contact, cytoskeletal remodelling, and apoptosis in the BA22 region. For the BA10 region, we observed the strongest changes in reproductive signalling, tissue remodelling, and cell differentiation. Further exploratory analysis also identified potentially disease-relevant processes that were undetected in our more stringent primary analysis, including autophagy in the BA22 region and the amyloid process in the BA10 region. Collectively, our analysis suggests disruption of many common pathways and processes underpinning synaptic plasticity in both regions in schizophrenia, whereas individual regions emphasize changes in certain pathways that may help to highlight pathway-specific therapeutic opportunities to treat negative or positive symptoms of the disease.

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The quantification of gene expression in an animal model of brain ischaemia using TaqMan™ real-time RT-PCR

Cited by 78 publications

References 38 publications

Induction of housekeeping gene expression after subarachnoid hemorrhage in dogs

Induction of housekeeping gene expression after subarachnoid hemorrhage in dogs

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

Transcription and pathway analysis of the superior temporal cortex and anterior prefrontal cortex in schizophrenia

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