Validation of Four Reference Genes for Quantitative mRNA Expression Studies in a Rat Model of Inflammatory Injury

Walder, Roxanne Y.; Wattiez, Arnaud; White, Stephanie R.; Prado, Blanca Marquez de; Hamity, Marta V.; Hammond, Donna L.

doi:10.1186/1744-8069-10-55

Cited by 19 publications

(23 citation statements)

References 32 publications

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“…Actb was used as a normalization gene in the RVM because it was stably expressed in all four treatment groups (data not shown), consistent with an earlier report from this laboratory (Walder et al, 2014). For subsequent determination of Tac1 expression in the PAG, levels of Tac1 were normalized to the geometric mean of Actb and Mapk6 calculated as ΔC T followed by transformation to linear scale as 2 –Δ C T for statistical analysis.…”

Section: Methodssupporting

confidence: 82%

“…For subsequent determination of Tac1 expression in the PAG, levels of Tac1 were normalized to the geometric mean of Actb and Mapk6 calculated as ΔC T followed by transformation to linear scale as 2 –Δ C T for statistical analysis. Actb and Mapk6 were previously validated as reference genes for the RVM (Walder et al, 2014) and confirmed here for the PAG (M values of 0.55 for stability of gene expression) in the CFA model of inflammatory injury.…”

Section: Methodssupporting

confidence: 75%

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Changes in the disposition of substance P in the rostral ventromedial medulla after inflammatory injury in the rat

et al. 2016

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This study examined whether peripheral inflammatory injury increases the levels or changes the disposition of substance P (SubP) in the rostral ventromedial medulla (RVM), which serves as a central relay in bulbospinal pathways of pain modulation. Enzyme immunoassay and reverse transcriptase quantitative polymerase chain reaction were used to measure SubP protein and transcript, respectively, in tissue homogenates prepared from the RVM and the periaqueductal gray and cuneiform nuclei of rats that had received an intraplantar injection of saline or complete Freund's adjuvant (CFA). Matrix Assisted Laser Desorption/Ionization Time of Flight analysis confirmed that the RVM does not contain hemokinin-1, which can confound measurements of SubP because it is recognized equally well by commercial antibodies for SubP. Levels of SubP protein in the RVM were unchanged four hours, four days and two weeks after injection of CFA. Tac1 transcripts were similarly unchanged in the RVM four days or two weeks after CFA. In contrast, the density of SubP immunoreactive processes in the RVM increased 2-fold within four hours and 2.7-fold four days after CFA injection; it was unchanged at two weeks. SubP-immunoreactive processes in the RVM include axon terminals of neurons located in the periaqueductal gray and cuneiform nucleus. Substance P content in homogenates of the periaqueductal gray and cuneiform nucleus was significantly increased four days after CFA, but not at four hours or two weeks. Tac1 transcripts in homogenates of these nuclei were unchanged four days and two weeks after CFA. These findings suggest that there is an increased mobilization of SubP within processes in the RVM shortly after injury accompanied by an increased synthesis of SubP in neurons that project to the RVM. These findings are consonant with the hypothesis that an increase in SubP release in the RVM contributes to the hyperalgesia that develops after peripheral inflammatory injury.

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Section: Methodssupporting

confidence: 82%

Section: Methodssupporting

confidence: 75%

Changes in the disposition of substance P in the rostral ventromedial medulla after inflammatory injury in the rat

et al. 2016

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“…In recent years, there are lots of studies demonstrating the suitability of HPRT as a reference gene for normalization of qPCR to investigate gene expressions in many fields, such as a rat model of inflammatory injury [32], human umbilical vein endothelial cells [33], the spared nerve injury model of neuropathic pain [34], and pelvic lymph node tissue from patients with prostate cancer [35]. Meanwhile, the present study identified HRPT as suitable as a reference gene for mRNA studies in intestinal GVHD as well.…”

Section: Discussionmentioning

confidence: 99%

Identification of Suitable Reference Genes for Normalization of Real-Time Quantitative Polymerase Chain Reaction in an Intestinal Graft-Versus-Host Disease Mouse Model

Li¹,

Qiao²,

Yang³

et al. 2015

Transplantation Proceedings

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“…; Walder et al . ). The total reaction volume was 10 μL and this included 5 μL of the supermix, primers, cDNA template and nuclease‐free water.…”

Section: Methodsmentioning

confidence: 97%

“…Beta-actin (ACTB), TATA box binding protein (TBP) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) were used as endogenous reference genes, and the primers used for amplification of these genes were PrimePCR Assay ACTB, PrimePCR Assay TBP and PrimePCR Assay HPRT1, respectively. The resulting three transcripts were selected for analysis based on a previous demonstration of their stable expression in the central nervous system (Valente et al 2014;Walder et al 2014). The total reaction volume was 10 μL and this included 5 μL of the supermix, primers, cDNA template and nuclease-free water.…”

Section: Choline Acetyltransferase (Chat) and Nkcc1 Immunohistochemistrymentioning

confidence: 99%

Repeated anodal trans‐spinal direct current stimulation results in long‐term reduction of spasticity in mice with spinal cord injury

Mekhael

Begum

Samaddar

et al. 2019

The Journal of Physiology

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Key points Spasticity is a disorder of muscle tone that is associated with lesions of the motor system. This condition involves an overactive spinal reflex loop that resists the passive lengthening of muscles. Previously, we established that application of anodal trans‐spinal direct current stimulation (a‐tsDCS) for short periods of time to anaesthetized mice sustaining a spinal cord injury leads to an instantaneous reduction of spasticity. However, the long‐term effects of repeated a‐tsDCS and its mechanism of action remained unknown. In the present study, a‐tsDCS was performed for 7 days and this was found to cause long‐term reduction in spasticity, increased rate‐dependent depression in spinal reflexes, and improved ground and skill locomotion. Pharmacological, molecular and cellular evidence further suggest that a novel mechanism involving Na‐K‐Cl cotransporter isoform 1 mediates the observed long‐term effects of repeated a‐tsDCS. Abstract Spasticity can cause pain, fatigue and sleep disturbances; restrict daily activities such as walking, sitting and bathing; and complicate rehabilitation efforts. Thus, spasticity negatively influences an individual's quality of life and novel therapeutic interventions are needed. We previously demonstrated in anaesthetized mice that a short period of trans‐spinal subthreshold direct current stimulation (tsDCS) reduces spasticity. In the present study, the long‐term effects of repeated tsDCS to attenuate abnormal muscle tone in awake female mice with spinal cord injuries were investigated. A motorized system was used to test velocity‐dependent ankle resistance and associated electromyographical activity. Analysis of ground and skill locomotion was also performed, with electrophysiological, molecular and cellular studies being conducted to reveal a potential underlying mechanism of action. A 4 week reduction in spasticity was associated with an increase in rate‐dependent depression of spinal reflexes, and ground and skill locomotion were improved following 7 days of anodal‐tsDCS (a‐tsDCS). Secondary molecular, cellular and pharmacological experiments further demonstrated that the expression of K‐Cl co‐transporter isoform 2 (KCC2) was not changed in animals with spasticity. However, Na‐K‐Cl cotransporter isoform 1 (NKCC1) was significantly up‐regulated in mice that exhibited spasticity. When mice were treated with a‐tsDCS, down regulation of NKCC1 was detected, and this level did not significantly differ from that in the non‐injured control mice. Thus, long lasting reduction of spasticity by a‐tsDCS via downregulation of NKCC1 may constitute a novel therapy for spasticity following spinal cord injury.

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Validation of Four Reference Genes for Quantitative mRNA Expression Studies in a Rat Model of Inflammatory Injury

Cited by 19 publications

References 32 publications

Changes in the disposition of substance P in the rostral ventromedial medulla after inflammatory injury in the rat

Changes in the disposition of substance P in the rostral ventromedial medulla after inflammatory injury in the rat

Identification of Suitable Reference Genes for Normalization of Real-Time Quantitative Polymerase Chain Reaction in an Intestinal Graft-Versus-Host Disease Mouse Model

Repeated anodal trans‐spinal direct current stimulation results in long‐term reduction of spasticity in mice with spinal cord injury

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