The use of quantitative RT-PCR to measure mRNA expression in a rat model of focal ischemia — caspase-3 as a case study

Harrison, David; Medhurst, Andrew D.; Bond, Brian; Campbell, C. A.; Davis, Robert P.; Philpott, Karen L.

doi:10.1016/s0169-328x(99)00305-8

Cited by 109 publications

(66 citation statements)

References 26 publications

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“…It appeared that this variance contributed to a failed detection of experimental effect, making b-actin a poor choice for that particular FPI experimental design as well. In this context, prior studies have also reported rapid upregulation of b-actin and GAPDH in rodent models of cerebral ischemia (Harrison et al, 2000;Kobayashi et al, 2004). Given that secondary ischemia is often a component of TBI pathogenesis, we would predict an acute 2-day increase in parietotemporal cortical b-actin transcript after FPI as was observed.…”

Section: Figmentioning

confidence: 57%

“…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. For example, GAPDH and cyclophilin A were both stably expressed during the first 24 h after middle cerebral artery occlusion in rats (Harrison et al, 2000). Furthermore, in the human post-mortem brain, optimal reference genes vary with disease state and often differ in choice ranking for a given brain region relative to those reported for animal models of brain injury (Coulson et al, 2008).…”

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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Section: Figmentioning

confidence: 57%

Section: Introductionmentioning

confidence: 99%

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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“…To obtain cDNA, 200 ng total RNA was reverse-transcribed with use of the Taqman Gold reverse transcription-polymerase chain reaction (RT-PCR) kit (Applied Biosystems). Real-time PCR analysis was done with an iCycler iQ detection system (Bio-Rad) in a master mix that contained specific primers, as described elsewhere (400 nmol/L for Ca v 1.2 21 and cyclophilin 22 ; 500 nmol/L for K v 4.2 23 ), AmpliTaq Gold DNA polymerase, and Taqman probes (100 nmol/L) tagged at the 5Ј end with the fluorescent molecule 6-carboxyfluorescein (5Ј-FAM) containing the fluorescent quencher moiety 6-carboxytetramethylrhodamine (3Ј-TAMRA) in 3Ј. Each measurement was performed in triplicate.…”

Section: Cell Isolation and Recording Techniquesmentioning

confidence: 99%

Mineralocorticoid Receptor Antagonism Prevents the Electrical Remodeling That Precedes Cellular Hypertrophy After Myocardial Infarction

et al. 2004

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Background-Cardiac hypertrophy underlies arrhythmias and sudden death, for which mineralocorticoid receptor (MR) activity has recently been implicated. We sought to establish the sequence of ionic events that link the initiating insult and MR to hypertrophy development. Methods and Results-Using whole-cell, patch-clamp and quantitative reverse transcription-polymerase chain reaction techniques on right ventricular myocytes of a myocardial infarction (MI) rat model, we examined the cellular response over time. One week after MI, no sign of cellular hypertrophy was found, but action potential duration (APD) was lengthened. Both an increase in Ca 2ϩ current (I Ca ) and a decrease in K ϩ transient outward current (I to ) underlay this effect. Consistently, the relative expression of mRNA coding for the Ca 2ϩ channel ␣1C subunit (Ca v 1.2) increased, and that of the K ϩ channel K v 4.2 subunit decreased. Three weeks after MI, AP prolongation endured, whereas cellular hypertrophy developed. I Ca density, Ca v 1.2, and K v 4.2 mRNA levels regained control levels, but I to density remained reduced. Long-term treatment with RU28318, an MR antagonist, prevented this electrical remodeling. In a different etiologic model of abdominal aortic constriction, we confirmed that APD prolongation and modifications of ionic currents precede cellular hypertrophy. Conclusions-Electrical remodeling, which is triggered at least in part by MR activation, is an initial, early cellular response to hypertrophic insults.

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“…An ABI PRISM 7700 Sequence Detection System (Centre for Gene Research, University of Otago) was used to detect fluorescence during each PCR cycle. The thermal cycling conditions were set at 50 C for 2 min and 95 C for 10 min initially, followed by 15 s at 95 C (melting step) and 1 min at 60 C (anneal/extend step) for 40 cycles (Harrison et al 2000). The initial steps were important to activate the AmpErase UNG enzyme, which removes potential contamination from previous TaqMan PCR reactions, and to activate the AmpliTaq Gold DNA polymerase present in the TaqMan Universal PCR Master Mix.…”

Section: Real-time Quantitative Pcrmentioning

confidence: 99%

Quantitation of prolactin receptor mRNA in the maternal rat brain during pregnancy and lactation

Augustine¹,

Kokay²,

Andrews³

et al. 2003

Journal of Molecular Endocrinology

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Prolactin receptor (PRL-R) expression in the brain is increased in lactating rats compared with non-pregnant animals. The aim of the present study was to determine the time-course of changes in PRL-R mRNA levels during pregnancy and/or lactation, and to determine relative levels of the two forms (short and/or long form) of receptor mRNA in specific brain regions. Brains were collected from female rats on dioestrus, days 7, 14 or 21 of pregnancy, day 7 of lactation or day 7 post-weaning. Frozen, coronal sections were cut (300 µm) and specific hypothalamic nuclei and the choroid plexus were microdissected using a punch technique. Total RNA was extracted and reverse transcribed, then first strand cDNA was amplified using quantitative real-time PCR. Results showed an up-regulation of long-form PRL-R mRNA in the choroid plexus by day 7 of pregnancy compared with dioestrus, which further increased on days 14 and 21 of pregnancy and day 7 of lactation, and then decreased to dioestrous levels on day 7 post-weaning. Short-form PRL-R mRNA levels increased on day 14 of pregnancy relative to dioestrus, increased further on day 7 of lactation and decreased on day 7 post-weaning. Changes in mRNA were reflected in increased levels of PRL-R immunoreactivity in the choroid plexus during pregnancy and lactation, compared with dioestrus. In the arcuate nucleus, long-form PRL-R mRNA was increased during pregnancy. In contrast to earlier work, no significant changes in short-or long-form PRL-R mRNA expression were detected in several other hypothalamic nuclei, suggesting that changes in hypothalamic mRNA levels may not be as marked as previously thought. The up-regulation of PRL-R mRNA and protein expression in the choroid plexus during pregnancy and lactation suggest a possible mechanism whereby increasing levels of peripheral prolactin during pregnancy may have access to the central nervous system. Together with expression of long-form PRL-R mRNA in specific hypothalamic nuclei, these results support a role for prolactin in regulating neuroendocrine and behavioural adaptations in the maternal brain.

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The use of quantitative RT-PCR to measure mRNA expression in a rat model of focal ischemia — caspase-3 as a case study

Cited by 109 publications

References 26 publications

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

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

Mineralocorticoid Receptor Antagonism Prevents the Electrical Remodeling That Precedes Cellular Hypertrophy After Myocardial Infarction

Quantitation of prolactin receptor mRNA in the maternal rat brain during pregnancy and lactation

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