Genomic DNA from control fibroblasts from patient 5 data from: Methylation profiling by genome tiling array

Experiment type: Methylation profiling by genome tiling array Summary: 3mm punch biopsies were taken from a healed normotrophic scar and a contralateral matched control site in burn patients with a scar at least 1 year old. Fibroblasts were cultured from explants to passage 2 and DNA was extracted and run on methylation arrays to examine differences in scar and control fibroblast gene expression Normotrophic scar maintains its abnormal scar phenotype for the rest of the patients life, long after the injury has healed. Differences in gene expression may reaveal target genes that can be modulated to improve scar appearance. Overall design: 6 patients had normotrophic scar and matched control fibroblasts extracted and compared. Sample type genomic Source name Patient 5 Control Organism Homo sapiens Characteristics Sex: Male age (years): 25 time since injury (years): 5 Treatment protocol There were no treatments applied to the scar and control cells in vitro. DNA was bisulfite treated prior to processing on the chip Growth protocol 3mm punch biopsies were taken from the forearm scar site and a matched site on the uninjured forearm by the attendant physician. Tissue was transported to the lab where it was placed in a petri dish and sliced into three equal sized potions. Pieces of the biopsy were then placed dermis side down in a T-25 (25 cm2) (Greiner Bio-One, Germany) culture flask without any media, and a drop of 100% foetal bovine serum (FBS) added to the surface of each piece. They were then cultured in DMEM with 10% FBS and 5% pen/strep until passage 2, at which point DNA and RNA was extracted. Extracted molecule genomic DNA Extraction protocol Genomic DNA was extracted using either the Promega Wizard SV Genomic DNA system (Patients 1 and 2, Promega, USA) or the QIAamp DNA mini kit system (Patients 3-6, Qiagen, Netherlands) as per manufacturer’s instructions Label Cy3 and Cy5 Label protocol Standard Illumina protocol Hybridization protocol bisulphite converted DNA was amplified, fragmented and hybridised to Illumina Infinium Human Methylation27 Beadchip using standard Illumina protocol Scan protocol Arrays were imaged using BeadArray Reader using standard recommended Illumina scanner setting Description Normal Fibroblast DNA Data processing Methylation data was imported from the raw files using Illumina® GenomeStudio and analysed using R statistics software, using the RnBeads package实验类型：基因组平铺阵列（genome tiling array）甲基化谱分析 摘要：从瘢痕形成至少1年的烧伤患者的成熟正常瘢痕（normotrophic scar）部位及对侧匹配对照部位获取3mm穿刺活检组织。将外植体培养的成纤维细胞传至第2代，提取DNA并进行甲基化芯片检测，以分析瘢痕与对照成纤维细胞的基因表达差异。成熟正常瘢痕会在患者余生中维持其异常瘢痕表型，即便损伤早已愈合。基因表达差异的分析可揭示可被调控以改善瘢痕外观的靶基因。 实验设计：对6名患者的成熟正常瘢痕及匹配对照成纤维细胞进行提取并开展对比分析。 样本类型：基因组样本 样本来源名称：患者5对照 物种：智人（Homo sapiens） 样本特征：性别：男性；年龄（岁）：25；损伤后时长（年）：5 处理方案：瘢痕与对照细胞未施加体外处理。DNA在芯片上机检测前进行亚硫酸氢盐转化处理。 培养方案：主治医师从前臂瘢痕部位及未损伤前臂的匹配位点获取3mm穿刺活检组织。组织被转运至实验室，置于培养皿中并切成三等份。将活检组织块以真皮面朝下的方式放置于T-25（25 cm²，德国Greiner Bio-One公司）培养瓶中，不添加培养基，并在每块组织表面滴加100%胎牛血清（FBS）。随后将组织置于添加10%胎牛血清与5%青霉素-链霉素（pen/strep）的达尔伯克改良伊格尔培养基（DMEM）中培养至第2代，此时提取DNA与RNA。 提取分子：基因组DNA（genomic DNA） 提取方案：按照制造商说明书，分别采用Promega Wizard SV基因组DNA提取试剂盒（患者1、2，美国Promega公司）或QIAamp DNA迷你提取试剂盒（患者3-6，荷兰Qiagen公司）提取基因组DNA。 标记染料：Cy3与Cy5 标记方案：采用标准Illumina标记流程。 杂交方案：将亚硫酸氢盐转化后的DNA进行扩增、片段化，并采用标准Illumina流程杂交至Illumina Infinium人类甲基化27K微珠芯片（Illumina Infinium Human Methylation27 Beadchip）。 扫描方案：使用微珠阵列扫描仪（BeadArray Reader）按照Illumina官方推荐的标准扫描参数对芯片进行成像。 样本描述：正常成纤维细胞DNA 数据处理：使用Illumina® GenomeStudio软件从原始文件中导入甲基化数据，并采用R统计软件及RnBeads包进行分析。