Genomic DNA from control fibroblasts from patient 3 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 3 Control Organism Homo sapiens Characteristics Sex: Male age (years): 19 time since injury (years): 2 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：全基因组平铺阵列甲基化谱分析 摘要：本研究纳入瘢痕形成至少1年的烧伤患者，从其愈合后的正常型瘢痕（normotrophic scar）及对侧匹配对照部位获取3mm钻孔活检组织。从组织外植体中培养成纤维细胞至第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。正常型瘢痕会在患者余生中持续维持其异常瘢痕表型，即便损伤已愈合许久。基因表达的差异或可揭示可被调控以改善瘢痕外观的靶基因。 实验设计：共纳入6名患者，分别提取其正常型瘢痕及匹配对照的成纤维细胞并进行比对分析。 样本类型：基因组样本 样本来源名称：患者3对照 物种：智人（Homo sapiens） 样本特征：性别：男；年龄（岁）：19；损伤后时间（年）：2 处理方案：体外实验中未对瘢痕及对照细胞施加任何处理。DNA在芯片检测前进行亚硫酸氢盐处理。 培养方案：主治医师从患者前臂瘢痕部位及未受伤前臂的匹配部位获取3mm钻孔活检组织。样本被运送至实验室后置于培养皿中，切割为三等份。将活检组织块真皮面朝下放置于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1滴100%胎牛血清（FBS）。随后将组织在添加10% FBS及5%青链霉素的DMEM培养基中培养至第2代，此时提取DNA与RNA。 提取分子：基因组DNA 提取方案：按照制造商说明书，分别使用Promega Wizard SV基因组DNA提取系统（患者1和2，美国Promega公司）或QIAamp DNA迷你试剂盒（患者3-6，荷兰Qiagen公司）提取基因组DNA。 标记物：Cy3与Cy5 标记流程：采用标准Illumina标记方案 杂交流程：将亚硫酸氢盐转化后的DNA进行扩增、片段化，随后按照标准Illumina方案杂交至Illumina Infinium Human Methylation27 Beadchip 扫描流程：使用BeadArray阅读器，按照Illumina官方推荐的标准扫描仪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软件从原始文件导入甲基化数据，并借助R统计软件及RnBeads包进行数据分析。