Strain: C57Bl/KS-db/+ Gender: female Age: 9-13 weeks old
Biomaterial provider
Mice were purchased from Møllegaard, Denmark. Wounding experiment and tissue collection was performed at the University of Tromsø, Norway
Treatment protocol
Operations: Wounds were established on the backs of mice under general anesthesia and after chemical depilation and desinfection with chlorhexidine, by excising skin and panniculus carnosus of 1.5x1.5 cm area. A semipermeable transparent polyurethane dressing Opsite FlexigridÒ (Smith & Nephew Medical Ltd., Hull, England) was fixed to the skin with tissue adhesive HistoacrylÒ (B. Braun Melsungen AG, Melsungen, Germany) and eight interrupted 5-0 MonosofÔ sutures (Auto Suture Company, Norwalk, CT, USA). Post-operative animals received analgesia right after, and following 12 hours after the surgical procedure. To provide sufficient hydration, the animals were given two subcutaneous injections of an isotonic electrolyte solution Ringer Acetate (Fresenius Kabi Norge AS, Halden, Norway) (dose: 0.030 – 0.050 ml/g body wt) immediately and at 2 hours post wounding. After the operation, the mice were put into a heating chamber at initial temperature 27 – 31° C, adjusted to 23 –25° C during awakening. Mice were killed by CO2-suffocation on either day 2, 6 or day 15 post-surgery. Samples were collected into microcentrifuge tubes with RNA-later, kept refrigerated overnight and then frozen at -70°C.
Extracted molecule
total RNA
Extraction protocol
RNA-later preserved wound and skin samples were weighed and homogenized with Ultra Turrex T8 homogenizer (IKA/TamroLab). Total RNA was isolated with SV Total RNA Isolation System (Promega GmbH, Germany, catalogue number: Z3100) and treated with DNAse I (Technical Manual No. 048 from Promega).
Label
Cy3
Label protocol
500ng total RNA was amplified and labeled with cyanine 3-CTP (PerkinElmer/NEN Life Sciences, catalogue number NEL580) using Agilent Low RNA Input Fluorescent Linear amplification kit (Agilent Technologies, Inc., CA, USA, catalogue number: 5184-3523) according to the manufacturers instructions. Clean-up reaction was performed with Qiagen Rneasy mini kit (Qiagen Nordic, Sweden).
Hybridization protocol
Fluorescent cRNA was hybridized using Pronto! Universal Hybridisation kit (Corning, catalogue number: 40026) in Hybridization Chambers (Corning, catalogue 2551) according to the manufacturer’s instructions.
Scan protocol
The slides were scanned using a GenePix 4000B microarray scanner (Axon Instruments, Westbury, NY, USA) at laser intensity and photomultiplier tube settings giving the best dynamic range for each chip. Image segmentation and feature extraction were done with the ImaGene 4.1 software (Biodiscovery, Marina Del Rey, USA).
Description
2mm wide skin from wound edge tissues collected at day 15 post-wounding.
Data processing
Replicated spots (n=8) were averaged to produce a single measurement per gene and chip, and log-transformed mean-values were normalized for signal intensity by dividing with median intensity. Proteoglycan-related genes were tested for differential expression over time and for differential expression between wound bed and wound edge using two-way ANOVA.
