Genomic DNA from control fibroblasts from patient 2 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 2 Control

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
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