Biological material from humans
Skin biopsies with a 4.0 mm diameter punch were taken from the upper thigh or upper arm of 97 patients, before and 4 weeks after therapy with a drug to treat high blood pressure, after local anesthesia with ethyl chloride spray. Biopsies were immediately snap frozen in liquid nitrogen and stored at -80°C until further processing.
The protocol has been approved by the Medical Ethical Committee of the Academic Medical Center in Amsterdam. The feasibility approvals have been obtained from all the participating centers. This trial is registered in the Netherlands Trial Register under number NTR1423. Enrollment began in March 2008 and in October 2009 230 patients have been enrolled.
Biological material from mice
Four 3 to 4 months old SKH1 hairless mice were used in this experiment: two were untreated controls and two were irradiated with 300 J/m2 UV B light. 24 hours after exposure, both treated and untreated mice were euthanized by cervical dislocation. This was followed directly by biopsy sampling from the back to prevent RNA decay. For one mouse, biopsies were also taken from the abdomen. Biopsies were immediately snap frozen in liquid nitrogen and stored at -80°C until further processing.
Biopsies using a 1.0 mm diameter punch were unpractical as part of the material remained inside the skin upon punching. The 1.0 and 1.5 mm biopsies were taken by punching through the folded skin resulting in two holes and the 2.0 and 2.5 mm ones by punching a half moon shape on folded skin resulting in one round hole.
The study was agreed upon by the institute's Experimental Animal Ethical Committee and carried out in accordance with national legislation.
RNA isolation
Per RNA isolation, one 1.5 ml tube was filled beforehand with 75-100 mg phase-lock gel heavy (5-Prime) and pelleted for 30 s at 12,000 × g. Single biopsies were pulverized to a fine powder with a standard liquid nitrogen pre-chilled mortar and pestle. This powder was transferred to a 1.5 ml tube with 300 μl Qiazol (Qiagen). The homogenate was vortexed vigorously for 15 s and subsequently shaken for 10 min on a REAX 2000 (Heidolph). After a quick spin-down 60 μl chloroform was added to the homogenate, vortexed for 15 s and kept at room temperature for 3 min. The partly separated mixture was transferred as a whole to a pre-prepared phase-lock gel heavy containing tube and centrifuged for 15 min at 12,000 × g. The aqueous phase was transferred to a new 1.5 ml tube. RNA was purified by column precipitation according to the RNeasy MinElute Cleanup Handbook (version 2007) - Appendix D: RNA Cleanup after Lysis and Homogenization with Qiazol Lysis Reagent (Qiagen). At the end of the procedure, the RNA was eluted in 14 μl nuclease-free water. A PDF file with the complete protocol is available in a convenient format in the Additional file 1.
Microarrays with Human Affymetrix platform
Gene expression was analyzed with Human Exon 1.0 ST Arrays in biopsies from all 97 patients. Sense-strand cDNA was generated from total RNA using Ambion WT Expression Kit (Applied Biosystems) conform the manufacturer's instructions. Further steps were performed using the manufacturer's protocols for the GeneChip platform (Affymetrix). Those included purification of double-stranded cDNA, synthesis of aRNA by in vitro transcription, recovery and quantification of biotin-labeled aRNA, fragmentation of this aRNA and subsequent hybridization to the microarray, post-hybridization washings and detection of the hybridized aRNA using a streptavidin-coupled fluorescent dye. Hybridized Affymetrix GeneChips were scanned using a GeneChip Scanner 3000-7G (Affymetrix). Image generation and feature extraction were performed using Affymetrix GCOS Software v1.4.0.036.
Microarrays with Mouse Roche NimbleGen platform
Gene expression of mouse samples was analyzed with a 12 × 135 k Mus musculus microarray (Catalog no. 05543797001, Design 090901 MM9 EXP HX12) containing 44,170 genes with 3 probes per target gene. Per RNA sample, 200 ng total RNA was amplified according to the Agilent LRILAK kit manual (Agilent technologies). Amino-allyl modified nucleotides were incorporated during the aRNA synthesis (2.5 mM rGAU (GE Healthcare), 0.75 mM rCTP (GE Healthcare), 0.75 mM AA-rCTP (TriLink Biotechnologies). Synthesized aRNA was purified with the E.Z.N.A. MicroElute RNA Clean Up Kit (Omega Bio-Tek). Test aRNA samples were labeled with Cy3 and a Reference sample (made by pooling equimolar amounts of RNA from Test samples) was labeled with Cy5. 5 μg of aRNA was dried down and dissolved in 50 mM carbonate buffer pH 8.5. Individual vials of Cy3/Cy5 from the mono-reactive dye packs (GE Healthcare) were dissolved in 200 μl DMSO. To each sample, 10 μl of the appropriate CyDye dissolved in DMSO was added and the mixture was incubated for 1 h. Reactions were quenched with the addition of 5 μl 4 M hydroxylamine (Sigma-Aldrich). The labeled aRNA was purified with the E.Z.N.A. MicroElute RNA Clean Up Kit. The yields of aRNA and CyDye incorporation were measured on the NanoDrop ND-1000.
Each hybridization mixture was made up from 1.1 μg Test (Cy3) and 1.1 μg Reference (Cy5) sample. Samples were dried and 1.98 μl of water was added. The hybridization cocktail was made according to the manufacturer's instructions (Roche NimbleGen Arrays User's Guide - Gene Expression Arrays Version 5.0, Roche NimbleGen). 5.22 μl from this mix was added to each sample. The samples were incubated for 5 min at 65°C and 5 min at 42°C prior to loading. Hybridization samples were loaded onto the microarrays, and hybridized for 18 hours at 42°C with the Roche NimbleGen Hybridization System 4. Afterwards, the slides were washed according to the Roche NimbleGen Arrays User's Guide - Gene Expression Arrays Version 5.0 and scanned in an ozone-free room with a DNA microarray scanner G2565CA (Agilent Technologies). Feature extraction was performed with NimbleScan v2.5 (Roche NimbleGen). The array data have been deposited in NCBI's Gene Expression Omnibus and is accessible through GEO Series accession number GSE28463 http://www.ncbi.nlm.nih.gov/geo/.
Data analysis
To generate the average log2 probe signal for the Affymetrix GeneChips, raw probe intensities without control probes were used. For the Nimblegen microarrays, raw sample channel data without control probes was used. Data handling, scatterplot generation and PCA analysis were performed using R-2.11.1 http://www.R-project.org and Bioconductor http://www.bioconductor.org/ software.