This protocol relies on the provision of human surgical tissue. Ethical approval and informed patient consent were obtained prior to experimentation, and the study conformed with the principles outlined in the Declaration of Helsinki.

1. Collection of tissue and ex vivo wounding

1. Collect surgical tissue following limb amputation/surgery into a sterile container containing Dulbecco's Modified Eagle Medium (DMEM) with 5% penicillin-streptomycin-fungizone, 2 mM L-glutamine and 10% fetal bovine serum (FBS; complete growth medium).
   NOTE: Tissue collected in this manner can be stored at 4 °C for up to 24 h prior to experimentation. To ensure viability of tissue for ex vivo experiments, it is recommended to process the tissue as soon as possible or to maintain a fixed time gap between collection and wounding to standardize experiments across multiple tissue donations. If the tissue is to be stored for any amount of time, an RNA stabilization solution should be added to the sample to maintain RNA integrity.
2. Using sterile forceps, transfer the tissue to a 60 mm Petri dish filled with sterile phosphate buffered saline (PBS) supplemented with 5% penicillin-streptomycin-fungizone. Trim the fat off the dermis using a sterile scalpel and/or surgical scissors and discard. Transfer the tissue to a fresh petri dish filled with sterile PBS.
3. Attach two sterile blades together such that there is a 1 mm gap between the two blades. Score two parallel linear lines, ~1 mm apart, across the length of the epidermis and the upper part of the papillary dermis^10,11.
4. Remove the epidermis between the score lines using sterile forceps and surgical scissors to create a linear wound.
5. Use a scalpel to cut the tissue into small rectangles (maximum size of 1 cm²) encompassing the wound with intact epidermis on either side.
   NOTE: To maintain tissue and subsequent RNA integrity, steps 1.2-1.5 should be conducted as quickly as possible.
6. Take an image of the wounded tissue using a microdissection microscope at 4x magnification ensuring the field of view captures the entirety of the wound and the surrounding intact epidermal tissue.
7. Using sterile forceps, place the tissue rectangles on a tissue culture insert in a 6-well plate and gently pipette 2 mL of complete growth medium into the well. This will ensure that the sample is cultured at the liquid-air interface.
8. Incubate at 37 °C in 5% CO₂ in air for up to 120 h.
   ​NOTE: The tissue should be imaged on a microdissection microscope at the same magnification as in step 1.6 at the end of the incubation. Images can also be taken every 24 h if necessary.

2. Tissue fixation and cryosectioning

1. Snap freeze the tissue in liquid nitrogen. Place a small amount of cryostat-compatible cutting medium onto a cryostat chuck and embed the tissue within it. Orient the tissue perpendicular to the chuck so that the cutting face will cut through all the layers of the skin including the wounded area. Store at -80 °C.
   NOTE: The protocol can be paused here.
2. Clean the cryostat at room temperature with 70% ethanol and RNase decontamination spray. Set the cryostat temperature to -28 °C.
3. Check that the orientation of chuck and cryostat blade can produce sections that encompass the full thickness of the tissue including the wounded tissue and underlying dermis.
4. Using the cryostat, cut up to ten 7 µm sections from each wound and place on to RNA-free microdissection slides.
5. Store the slides in the original box that the microdissections slides were provided in at -80 °C to ensure an RNase-free environment.
   ​NOTE: The protocol can be paused here but be aware RNA degradation will increase the longer the storage time is.

3. Laser capture microdissection

1. Place the microdissection slides with the tissue section in dry ice and go to the laser capture microdissection microscope room.
2. Air-dry the 1^st slide and quickly proceed to stain the sections with hematoxylin using an RNase-free hematoxylin and eosin staining kit immediately prior to performing laser capture microdissection.
3. Visualize the wounded tissue using a laser capture microdissection microscope at 10x magnification and take a picture of the area of interest that will be laser captured.
4. Trace around that area (for example, epithelial tongue, granulation tissue, microvessels) and collect into microdissection 0.5 mL diffuser isolation caps using the software instructions for the particular microscope that is being used.
5. Re-visualize and image the laser captured area of interest using the laser capture microdissection microscope at 10x magnification to demonstrate the location and complete excision of the dissected tissue.
   NOTE: Perform laser capture for each sample (up to 10 sections) for a maximum time of 1 h to minimize RNA degradation.
6. Add RNA storage buffer to the tube containing the microdissected tissue according to manufacturer's instructions, and store in dry ice until it can be transferred to a -80^°C freezer.
   ​NOTE: The protocol can be temporarily paused here.

4. Quantification of differential gene expression

1. Centrifuge the sample tubes at full speed for 1 min.
2. Isolate RNA from the microdissected tissue following the manufacturer's instructions.
3. Elute the RNA in a final volume of 12 µL of RNase free water and amplify the RNA using an RNA amplification kit and thermal cycler, according to manufacturer's instructions. Two rounds of amplification are recommended to ensure sufficient RNA for further analysis. Use the amplification conditions in Table 1.

Table 1: Amplification conditions.

4. Quantify the concentration and purity of the amplified RNA. Absorbance ratios of A260/230 (purity) and A260/280 (contaminants) > 1.8 are suitable for further analysis.
   NOTE: Purified amplified RNA can be used for focused gene expression studies (steps 4.5-4.6) or can be sent away for microarray analysis.
5. Synthesize cDNA using high-capacity cDNA reverse transcription kit according to manufacturer's instructions. Representative conditions are as follows:
   • 25 °C for 10 min
   • 37 °C for 2 h
   • 85 °C for 5 min
   • 4 °C hold
6. Perform quantitative PCR using 0.5 µL of cDNA and 1 µM primers (of the gene of interest) according to manufacturer's instructions. Representative conditions and primer sequences are as follows.
   1. Use the following conditions for quantitative PCR conditions: 95 °C for 10 minutes; 40 cycles of 95°C for 15 s and 62°C for 1 min; 95 °C for 5 min; and 4°C hold.
   2. Use the following primer sequences:
      GAPDH
      Forward    5'-TATAAATTGAGCCCGCAGCC-3'
      Reverse    5'-CGACCAAATCCGTTGACTCC-3'
      
      KRT17
      Forward    5'-AGGGAGAGGATGCCCACCTG-3'
      Reverse    5'-GCGGGAGGAGATGACCTTGC-3'

5. Data interpretation

1. Calculate the rate of wound healing using the images of the ex vivo tissue generated in steps 1.6-1.8 using ImageJ. There are multiple recent publications highlighting different variations on how to automatically analyze wound areas in ImageJ^12,13,14.
2. Quantify gene expression by using the ΔC_T method, comparing the threshold cycles for the gene of interest compared to that of the housekeeper (e.g., glyceraldehyde-3-phosphate; GAPDH). To do this, note the C_T value for the housekeeper and for the gene of interest.
   1. Calculate the difference between the two C_T values to normalize the amount of mRNA present and control for differences in RNA extraction concentrations between different isolations:
      ΔC_T = C_T (gene of interest) – C_T (housekeeper)
   2. Calculate the magnitude of difference between the C_T values to give the relative quantification of the gene of interest, expressed as a percentage of the housekeeper:
      ​Relative quantification = (2^-ΔCT) x 100
   3. Examine differences between different patient donors/disease states by comparing the relative quantifications of the genes of interest for each sample.
      NOTE: A schematic of the entire technique can be found in Figure 1.