Genomic DNA from normotrophic scar 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 Scar
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 Normotrophic Scar 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：基于基因组平铺阵列（genome tiling array）的甲基化谱分析

研究摘要：招募瘢痕形成至少1年的烧伤患者，分别于其愈合后正常增生性瘢痕（normotrophic scar）部位及对侧匹配对照部位采集3mm穿刺活检标本。从组织外植体中培养至第2代的成纤维细胞，提取基因组DNA并开展甲基化芯片检测，以分析瘢痕与对照成纤维细胞的基因表达差异。正常增生性瘢痕会在患者余生中持续维持异常瘢痕表型，即便损伤已愈合多年。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，分别提取其正常增生性瘢痕及匹配对照部位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号瘢痕组织

物种：智人（Homo sapiens）

样本特征：性别：男性；年龄（岁）：19；损伤后时间（年）：2

处理方案：瘢痕及对照细胞未施加任何体外处理。芯片检测前，对DNA进行亚硫酸氢盐（bisulfite）处理。

培养方案：主治医师从患者前臂瘢痕部位及未损伤前臂的匹配部位采集3mm穿刺活检标本。标本转运至实验室后，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（25cm²，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）。随后将细胞置于含10%胎牛血清及5%青链霉素（pen/strep）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，患者1、2的基因组DNA采用Promega Wizard SV基因组DNA提取试剂盒（美国Promega公司）提取，患者3-6的样本采用QIAamp DNA迷你试剂盒（荷兰Qiagen公司）提取。

标记：Cy3与Cy5

标记流程：采用标准Illumina标记方案

杂交流程：将经亚硫酸氢盐转化的DNA进行扩增、片段化，随后按照标准Illumina操作流程，杂交至Illumina Infinium人类甲基化27微珠芯片（Human Methylation27 Beadchip）。

扫描流程：使用BeadArray Reader扫描仪，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：正常增生性瘢痕成纤维细胞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包完成数据分析。