Genomic DNA from normotrophic scar fibroblasts from patient 6 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 6 Scar
Organism Homo sapiens
Characteristics Sex: Male
age (years): 29
time since injury (years): 3
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：基因组平铺芯片（genome tiling array）甲基化谱分析

摘要：本研究从瘢痕形成至少1年的烧伤患者体内，分别采集愈合后正常型瘢痕及对侧匹配对照位点的3mm穿刺活检标本。将外植体培养的成纤维细胞传至第2代，提取DNA并开展甲基化芯片检测，以对比分析瘢痕组与对照组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，即便损伤早已愈合。基因表达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，分别提取其瘢痕组织及匹配对照位点的成纤维细胞并进行对比分析。

样本类型：基因组样本
样本来源：患者6号瘢痕组织
受试生物：智人（Homo sapiens）
样本特征：性别：男性；年龄（岁）：29；伤后时长（年）：3
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在芯片检测前先进行亚硫酸氢盐转化处理。
培养方案：由接诊医师从患者前臂瘢痕部位及未受伤前臂的匹配位点获取3mm穿刺活检标本。标本运送至实验室后，置于培养皿中并切割为三等份。将活检组织块的真皮面朝下放置于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100%胎牛血清（FBS）。随后将组织置于添加10% FBS与5%青链霉素的杜尔贝科改良伊格尔培养基（DMEM）中培养至第2代，此时提取DNA与RNA。
提取分子：基因组DNA
提取方案：按照生产商说明书，分别使用Promega Wizard SV基因组DNA提取试剂盒（1、2号患者，美国Promega公司）或QIAamp DNA迷你提取试剂盒（3-6号患者，荷兰Qiagen公司）提取基因组DNA。
标记物：Cy3与Cy5
标记流程：采用Illumina标准操作流程。
杂交方案：按照Illumina标准流程，将经亚硫酸氢盐转化的DNA进行扩增、片段化后，与Illumina Infinium人类甲基化27微珠芯片（Illumina Infinium Human Methylation27 Beadchip）进行杂交。
扫描方案：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：正常型瘢痕成纤维细胞DNA
数据分析：从原始文件中导入甲基化数据，使用Illumina® GenomeStudio软件进行初步处理，并采用R统计软件及RnBeads包开展后续分析。