Amnion Tissue
In this project, I also with amnion tissue collection, ChIP preparation and Real-time PCR technique. The aim of this work was to determine the gene activating H3K4me3 and gene repressing H3K27me3 levels and their ratios at the promoter regions of NAMPT inflammatory gene.
Collection of Tissue
Fetal membranes were collected from women undergoing elective cesarean section at absence of labour [not in labour (NIL), n = 5], during active labour [in labour (IL), n = 4] and preterm (n=5). Reflected amnion was separated from the placenta within 30 min of delivery and processed immediately for ChIP or tissue incubation as required. Informed consent to donate tissues was obtained from women at the John Hunter Hospital in Newcastle, Australia, under approval from the Hunter New England Health Human Ethics Committee and the University of Newcastle Human Ethics Committee. Women with a history of infection, treated with nonsteroidal anti-inflammatory drugs, diagnosed with chorioamnionitis or severe asthma were excluded from the study.
Tissue extraction for ChIP
Amnion membranes were isolated as described elsewhere [1]. One gram tissue was fixed in phosphate-buffered saline (PBS) containing 1% formaldehyde for 20 min at room temperature. The tissues were washed in cold PBS containing 125 mM glycine and homogenized in 20 ml of swelling …show more content…
Promoter binding of H3K4me3 and H3K27me3 were calculated as percent recovery of input over the IgG negative control. Data were tested for distribution, transformed to approximate normal distribution and homogeneity of variance, and subjected to ANOVA (with repeated measures). Pairwise comparisons were performed using paired t-tests. In all statistical analyses, P < 0.05 was considered significant. Analyses were carried out using STATA version 8.0 (College Station,
In this project, I also with amnion tissue collection, ChIP preparation and Real-time PCR technique. The aim of this work was to determine the gene activating H3K4me3 and gene repressing H3K27me3 levels and their ratios at the promoter regions of NAMPT inflammatory gene.
Collection of Tissue
Fetal membranes were collected from women undergoing elective cesarean section at absence of labour [not in labour (NIL), n = 5], during active labour [in labour (IL), n = 4] and preterm (n=5). Reflected amnion was separated from the placenta within 30 min of delivery and processed immediately for ChIP or tissue incubation as required. Informed consent to donate tissues was obtained from women at the John Hunter Hospital in Newcastle, Australia, under approval from the Hunter New England Health Human Ethics Committee and the University of Newcastle Human Ethics Committee. Women with a history of infection, treated with nonsteroidal anti-inflammatory drugs, diagnosed with chorioamnionitis or severe asthma were excluded from the study.
Tissue extraction for ChIP
Amnion membranes were isolated as described elsewhere [1]. One gram tissue was fixed in phosphate-buffered saline (PBS) containing 1% formaldehyde for 20 min at room temperature. The tissues were washed in cold PBS containing 125 mM glycine and homogenized in 20 ml of swelling …show more content…
Promoter binding of H3K4me3 and H3K27me3 were calculated as percent recovery of input over the IgG negative control. Data were tested for distribution, transformed to approximate normal distribution and homogeneity of variance, and subjected to ANOVA (with repeated measures). Pairwise comparisons were performed using paired t-tests. In all statistical analyses, P < 0.05 was considered significant. Analyses were carried out using STATA version 8.0 (College Station,