RT quantitative PCR analysis of Rosveratrol plus N-acetylcysteine of oxidative stress and inflammation genes in rat choclea.
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ABSTRACT: Two month-old male Wistar rats received either resveratrol (10 mg/kg) plus N-acetylcysteine (400 mg/kg) or vehicle during 5 consecutive days. The second day of treatment, a concentrated solution of kanamycin and furosemide was placed on the round window of the right ears of rats receiving treatment or vehicle. Hearing was assessed by recording auditory brainstem responses before and 5, 16 and 23 days after starting the treatment with resveratrol and N-acetylcysteine. Cochlear samples were taken at the end of the treatment (5 days) and once the treatment had been suspended (23 days) to study cochlear cytoarchitecture and the evolution of the expression of oxidative stress, antioxidant defense and inflammation genes by using PCR arrays and RT-qPCR. qPCR gene expression profiling. Cochlea samples from 3-6 animals per group were analised. The expression of genes related to oxidative stress, antioxidant defense, and inflammation was studied at 5 and 23 days after the initiation of the 5 days treatment with resveratrol plus NAC.
Project description:Two month-old male Wistar rats received either resveratrol (10 mg/kg) plus N-acetylcysteine (400 mg/kg) or vehicle during 5 consecutive days. The second day of treatment, a concentrated solution of kanamycin and furosemide was placed on the round window of the right ears of rats receiving treatment or vehicle. Hearing was assessed by recording auditory brainstem responses before and 5, 16 and 23 days after starting the treatment with resveratrol and N-acetylcysteine. Cochlear samples were taken at the end of the treatment (5 days) and once the treatment had been suspended (23 days) to study cochlear cytoarchitecture and the evolution of the expression of oxidative stress, antioxidant defense and inflammation genes by using PCR arrays and RT-qPCR.
Project description:This study evaluated changes in gene expression upon IL-7 treatment in human naive and memory Treg. CD4+CD25+CD127low naïve and memory Treg were isolated from fresh PBMC and separately stimulated with ?CD3?/CD28 coupled beads (Invitrogen-Dynal) at a 1:10 bead/T cell ratio, treated with or without 10 ng/ml of rhIL-7 (R&D Systems) for 16 hours. qPCR gene expression profiling of CD4+CD25+CD127low naïve and memory Treg obtained from 2 separate donors. Cell lysates were prepared separately from the 2 donors and pooled prior to RNA extraction.
Project description:Ischemic preconditioning (4 cycles of 5 min ischemia and 5 min reperfusion) with a final reperfusion time of 120 minutes was performed in C57BL/6N (The Jackson Laboratory) or Per2-/- mice. Heart tissue was snap-frozen with clamps pre-cooled to the temperature of liquid nitrogen. Micro RNA was isolated with Trizol (Invitrogen) and purified using RT2 qPCR-Grade miRNA Isolation Kit (SABiosciences-Qiagen). cDNA template was generated using RT2 miRNA First Strand Kit (SABiosciences-Qiagen). miRNA expression was performed using RT2 miRNA PCR Array Mouse miFinder (SABiosciences-Qiagen).
Project description:Normal fibroblasts and SSc fibroblasts between the third and six subpassages were used for experiments. Total RNA was extracted from culture cells with ISOGEN (Nippon Gene, Tokyo, Japan). MicroRNA isolation from total RNA was performed using RT2 qPCR-Grade miRNA Isolation Kit (SA Bioscience). For RT2 Profiler PCR Array (SABioscience), microRNAs were reverse-transcribed into first strand cDNA using RT2 miRNA First Strand Kit (SABiosciences). A mixture of equal amounts of cDNAs from 5 normal fibroblasts or 5 SSc fibroblasts was prepared. The cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix and the mixture was added into a 96-well RT2 miRNA PCR Array (SABiosciences) that included primer pairs for 88 human microRNAs. Human dermal fibroblasts were obtained by skin biopsy from the affected areas (dorsal forearm) of 5 patients with diffuse cutaneous SSc and <2 years of skin thickening. Control fibroblasts were obtained by skin biopsies from 5 healthy donors. Control donors were each matched with a SSc patient for age, sex, and biopsy site.
Project description:Normal fibroblasts and SSc fibroblasts between the third and six subpassages were used for experiments. Normal and scleroderma fibroblasts were serum-starved for 24 hours and incubated in the presence or absence of TGF-β1 (2ng/ml) for 6 hours. Total RNA was extracted from culture cells with ISOGEN (Nippon Gene, Tokyo, Japan). MicroRNA isolation from total RNA was performed using RT2 qPCR-Grade miRNA Isolation Kit (SA Bioscience). For RT2 Profiler PCR Array (SABioscience), microRNAs were reverse-transcribed into first strand cDNA using RT2 miRNA First Strand Kit (SABiosciences). A mixture of equal amounts of cDNAs from 5 normal fibroblasts or 5 SSc fibroblasts was prepared. The cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix and the mixture was added into a 96-well RT2 miRNA PCR Array (SABiosciences) that included primer pairs for 88 human microRNAs. Human dermal fibroblasts were obtained by skin biopsy from the affected areas (dorsal forearm) of 5 patients with diffuse cutaneous SSc and <2 years of skin thickening. Control fibroblasts were obtained by skin biopsies from 5 healthy donors. Control donors were each matched with a SSc patient for age, sex, and biopsy site.
Project description:Skin specimens were derived from involved skin of 3 systemic scleroderma (SSc), 3 localized scleroderma (LSc) and 3 keloid patients. These skin samples and 3 control skins were collected and fixed in formaldehyde immediately after resections. The microRNA (miRNA) isolation from human skin tissue was performed using miRNeasy FFPE kit (Qiagen). For PCR array, miRNAs were reverse-transcribed into first strand cDNA using RT2 miRNA First Strand Kit (SABiosciences). A mixture of equal amounts of miRNAs from 3 normal skins, 3 SSc, 3 LSc or 3 keloid were prepared, and miRNA expression profile in each disease in vivo was evaluated using RT2 Profiler PCR Array. The cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix and the mixture was added into 96-well RT2 miRNA PCR Array that includes primer pairs for 88 human miRNAs (SABiosciences). Skin specimens were derived from involved skin of 3 systemic scleroderma (SSc), 3 localized scleroderma (LSc) and 3 keloid patients. These skin samples and 3 control skins were collected and fixed in formaldehyde immediately after resections. Control donors were each matched with diseases for age, sex, and biopsy site.
Project description:Activation of non-canonical TGF-β1 signaling indicates an autoimmune mechanism for bone marrow fibrosis in primary myelofibrosis Two condition experiment. Biological replicates: 3 control human healty subjects, 3 PMF patients
Project description:Activation of non-canonical TGF-?1 signaling indicates an autoimmune mechanism for bone marrow fibrosis in primary myelofibrosis Two condition experiment. Biological replicates: 3 control human healty subjects, 6 PMF patients
Project description:TGF-M-NM-21 signaling pathway of spleen of the Gata1low mouse model of myelofibrosis Four condition experiment. Biological replicates: 3 control CD1 mice, 3 Gata1low mice, 3 SB431542-treated Gata1low mice and 3 Vehicle-treated Gata1low mice.
Project description:TGF-M-NM-21 signaling pathway of bone marrow of the Gata1low mouse model of myelofibrosis Four condition experiment. Biological replicates: 3 control CD1 mice, 3 Gata1low mice, 3 SB431542-treated Gata1low mice and 3 Vehicle-treated Gata1low mice.