subject id: 2017 subject age: 41 number of treatments: 4 time: day 112 tissue: skin biopsy
Treatment protocol
untreated or treated with Fraxel® laser set to 12mj and 250mtz
Growth protocol
This study was approved by the Schulman Associates Institutional Review Board. All subjects provided written informed consent prior to undergoing any study procedures. Fourteen women aged 30-55 with Fitzpatrick skin type I-IV were enrolled in this study. Fractional laser treatments were performed using a Fraxel® laser set to 12mj and 250mtz with a 1 cm2 treatment area, with all treatments and biopsy sampling performed by licensed medical personnel. For this study, the 14 study participants were treated on the back at 9 different equally spaced sites between the neck, upper buttocks and the left/right edges of the back with a minimum distance of 5 cm between each treatment site (plus one untreated site), and all sites were tattooed for site recognition. Sixty minutes prior to Fraxel® laser treatment, the treatment site was dosed with a topical anesthetic (BLT Cream: 20% benzocaine, 6% lidocaine, 4% tetracaine) after which the hand-held Fraxel® laser was moved over the treatment site 8 times. The treatment groups were as follows: (1) single laser treatments at 1, 3, 7, 14, 21 and 28 days before biopsy and (2) multiple laser treatments at 28-day intervals with 1, 2, 3, and 4 treatments. Following the final treatment, a 4 mm full thickness biopsy was taken at each site (including a non-treated site), the biopsy site was sutured, and the biopsy sample was placed in a cryotube and flash frozen in liquid nitrogen.
Extracted molecule
total RNA
Extraction protocol
Flash-frozen, bisected 4mm full-thickness skin punch biopsies were homogenized in TRIzol reagent (ThermoFisher, Waltham, MA) and total RNA was extracted according to the manufacturer's protocol. RNA was further purified using RNEasy spin columns (QIAGEN, Hilden, Germany).
Label
biotin
Label protocol
Biotinylated cRNA were prepared using the Affymetrix HT 3’ IVT Express kit (Cat. #901253) on a Beckman Biomek FXp Laboratory Automation Workstation (Beckman Cat. #A31842).
Hybridization protocol
Biotinylated cRNA was fragmented by limited alkaline hydrolysis and then hybridized overnight to Affymetrix GeneTitan U219 microarray plates using the Affymetrix GeneTitan instrument
Scan protocol
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
Data processing
The PLIER algorithm, quantile normalization, was performed in R as the processing protocol to normalize the data for subsequent analysis.
