Rat pro-inflammatory cytokine and cytokine related mRNA quantification by real-time polymerase chain reaction using SYBR green

Peinnequin, André; Mouret, Catherine; Birot, Olivier; Alonso, Antonia; Mathieu, Jacques; Clarençon, Didier; Agay, Diane; Chancerelle, Yves; Multon, E.

doi:10.1186/1471-2172-5-3

Cited by 265 publications

(67 citation statements)

References 27 publications

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“…Gene expressions were analyzed by real-time PCR using the SYBR Green PCR Master Mix [40] (Applied Biosystems, Darmstadt, Germany) with the ABI PRISM 7900 Sequence Detection System. GAPDH was used as housekeeping gene.…”

Section: Methodsmentioning

confidence: 99%

Hydroxyfasudil-Mediated Inhibition of ROCK1 and ROCK2 Improves Kidney Function in Rat Renal Acute Ischemia-Reperfusion Injury

et al. 2011

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Renal ischemia-reperfusion (IR) injury (IRI) is a common and important trigger of acute renal injury (AKI). It is inevitably linked to transplantation. Involving both, the innate and the adaptive immune response, IRI causes subsequent sterile inflammation. Attraction to and transmigration of immune cells into the interstitium is associated with increased vascular permeability and loss of endothelial and tubular epithelial cell integrity. Considering the important role of cytoskeletal reorganization, mainly regulated by RhoGTPases, in the development of IRI we hypothesized that a preventive, selective inhibition of the Rho effector Rho-associated coiled coil containing protein kinase (ROCK) by hydroxyfasudil may improve renal IRI outcome. Using an IRI-based animal model of AKI in male Sprague Dawley rats, animals treated with hydroxyfasudil showed reduced proteinuria and polyuria as well as increased urine osmolarity when compared with sham-treated animals. In addition, renal perfusion (as assessed by 18F-fluoride Positron Emission Tomography (PET)), creatinine- and urea-clearances improved significantly. Moreover, endothelial leakage and renal inflammation was significantly reduced as determined by histology, 18F-fluordesoxyglucose-microautoradiography, Evans Blue, and real-time PCR analysis. We conclude from our study that ROCK-inhibition by hydroxyfasudil significantly improves kidney function in a rat model of acute renal IRI and is therefore a potential new therapeutic option in humans.

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Section: Methodsmentioning

confidence: 99%

Hydroxyfasudil-Mediated Inhibition of ROCK1 and ROCK2 Improves Kidney Function in Rat Renal Acute Ischemia-Reperfusion Injury

et al. 2011

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“…Relationship between the C T and the logarithm of the cDNA concentration were studied according to (1) the correlation coefficient (r) and (2) the amplification efficiency. Correlation coefficients confirm the linearity and PCR efficiency (Ex) was calculated using the equation Ex 5 (10 21/slope ) 2 1 3 100 (Peinnequin et al, 2004). The linearity and efficiency of amplification of PCR assays among different templates allowed an accurate quantification of the different genes (Table 1).…”

Section: Determination Of Mrna Of Myelin Proteins Andmentioning

confidence: 99%

Effects of progesterone on oligodendrocyte progenitors, oligodendrocyte transcription factors, and myelin proteins following spinal cord injury

Labombarda

González

Lima

et al. 2008

Glia

101

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Progesterone is emerging as a myelinizing factor for central nervous system injury. Successful remyelination requires proliferation and differentiation of oligodendrocyte precursor cells (OPC) into myelinating oligodendrocytes, but this process is incomplete following injury. To study progesterone actions on remyelination, we administered progesterone (16 mg/kg/day) to rats with complete spinal cord injury. Rats were euthanized 3 or 21 days after steroid treatment. Short progesterone treatment (a) increased the number of OPC without effect on the injury-induced reduction of mature oligodendrocytes, (b) increased mRNA and protein expression for the myelin basic protein (MBP) without effects on proteolipid protein (PLP) or myelin oligodendrocyte glycoprotein (MOG), and (c) increased the mRNA for Olig2 and Nkx2.2 transcription factors involved in specification and differentiation of the oligodendrocyte lineage. Furthermore, long progesterone treatment (a) reduced OPC with a concomitant increase of oligodendrocytes; (b) promoted differentiation of cells that incorporated bromodeoxyuridine, early after injury, into mature oligodendrocytes; (c) increased mRNA and protein expression of PLP without effects on MBP or MOG; and (d) increased mRNA for the Olig1 transcription factor involved in myelin repair. These results suggest that early progesterone treatment enhanced the density of OPC and induced their differentiation into mature oligodendrocytes by increasing the expression of Olig2 and Nkx2.2. Twenty-one days after injury, progesterone favors remyelination by increasing Olig1 (involved in repair of demyelinated lesions), PLP expression, and enhancing oligodendrocytes maturation. Thus, progesterone effects on oligodendrogenesis and myelin proteins may constitute fundamental steps for repairing traumatic injury inflicted to the spinal cord.

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“…RNA (3 µg) was reverse-transcribed into single-stranded cDNA (Maxima First Strand cDNA Synthesis Kit, Thermo Fisher Scientific Inc.), and RT-PCR was performed (Maxima SYBR Green qPCR Master Mix, Thermo Fisher Scientific Inc.) using the respective primers for CYP isoforms: CYP1A2, CYP2B1, CYP2B2, CYP3A1, CYP2C6, CYP2C11, CYP2C13 (Table 1). The quantity of the target mRNA relative to that of the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) was determined [28]. …”

Section: Methodsmentioning

confidence: 99%

Isoform-Dependent Changes in Cytochrome P450-Mediated Drug Metabolism after Portal Vein Ligation in the Rat

et al. 2018

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Background: Surgical removal of complicated liver tumors may be realized in two stages via selective portal vein ligation, inducing the atrophy of portally ligated lobes and the compensatory hypertrophy of nonligated liver lobes. Unlike morphological changes, functional aspects such as hepatic cytochrome P450 (CYP)-mediated drug metabolism remain vaguely understood, despite its critical role in both drug biotransformation and hepatic functional analysis. Our goal was the multilevel characterization of hepatic CYP-mediated drug metabolism after portal vein ligation in the rat. Methods: Male Wistar rats (n = 24, 210–230 g) were analyzed either untreated (controls; n = 4) or 24/48/72/168/336 h (n = 4 each) following portal vein ligation affecting approximately 80% of the liver parenchyma. Besides the weights of ligated and nonligated lobes, pentobarbital (30 mg/kg)-induced sleeping time, CYP1A(2), CYP 2B(1/2), CYP2C(6/11/13), CYP3A(1) enzyme activities, and corresponding isoform mRNA expressions, as well as CYP3A1 protein expression were determined by in vivo sleeping test, CYP isoform-selective assays, polymerase chain reaction, and immunohistochemistry, respectively. Results: Portal vein ligation triggered atrophy in ligated lobes and hypertrophy nonligated lobes. Sleeping time was transiently elevated (p = 0.0451). After an initial rise, CYP1A, CYP2B, and CYP3A enzyme activities dropped until 72 h, followed by a potent increase only in the nonligated lobes, paralleled by an early (24–48 h) transcriptional activation only in nonligated lobes. CYP2C enzyme activities and mRNA levels were bilaterally rapidly decreased, showing a late reconvergence only in nonligated lobes. CYP3A1 immunohistochemistry indicated substantial differences in positivity in the early period. Conclusions: Beyond the atrophy-hypertrophy complex, portal vein ligation generated a transient suppression of global and regional drug metabolism, re-established by an adaptive, CYP isoform-dependent transcriptional response of the nonligated lobes.

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Rat pro-inflammatory cytokine and cytokine related mRNA quantification by real-time polymerase chain reaction using SYBR green

Cited by 265 publications

References 27 publications

Hydroxyfasudil-Mediated Inhibition of ROCK1 and ROCK2 Improves Kidney Function in Rat Renal Acute Ischemia-Reperfusion Injury

Hydroxyfasudil-Mediated Inhibition of ROCK1 and ROCK2 Improves Kidney Function in Rat Renal Acute Ischemia-Reperfusion Injury

Effects of progesterone on oligodendrocyte progenitors, oligodendrocyte transcription factors, and myelin proteins following spinal cord injury

Isoform-Dependent Changes in Cytochrome P450-Mediated Drug Metabolism after Portal Vein Ligation in the Rat

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