Title: Affymetrix CEL analysis. Description:
This document describes Affymetrix recommended hybridization protocols for use with GeneChip Exon Arrays.
Exons of the Core Meta-Probeset were used. Quantile Normalisation and RMA background correction was performed. Data analysis performed with Partek Genomics Suite, version 6.3, Copyright 2007, Partek Inc., St. Charles, MO, USA.
Samples were prepared using WT-Ovation™ Exon and Encore™ Biotin modules (NuGEN™ Technologies Inc)
(Parameters: Amplification = OTHER: Ovation™ Pico WTA amplification system (NuGEN™ Technologies Inc., San Carlos, Ca), Mass unit = Micro gram)
Human scalp skin was obtained following informed consent, in accordance with ethical approval from the North West Research Ethics Committee, UK (REC Ref. 07/H1010/69) or institutional review board approval from the University of Lübeck, Germany. Adult LPP patients (mean age = 59.4 years; range 23 – 73; Suppl Table A) were recruited and biopsied. A diagnosis of Lichen planopilaris (LPP) was established both clinically and histopathologically, following generally accepted criteria (Harries, M. J., R. M. Trueb, et al. (2009). “How not to get scar(r)ed: pointers to the correct diagnosis in patients with suspected primary cicatricial alopecia.” Br J Dermatol 160(3): 482-501). Lesional samples (L) were obtained from clinically inflamed edges of alopecia containing a reduced density of hairs. Non-lesional samples (U) were taken from LPP patient scalp skin that did not show lesions. Samples were snap-frozen.
(Parameters: time unit = seconds, temperature unit = C)
Extraction of RNA was performed using spin-column technology following the manufacturer’s protocol (RNeasy® Micro Kit, Qiagen® Ltd, Crawley, Sussex). Whole transcriptome amplification using the Ovation™ Pico WTA amplification system (NuGEN™ Technologies Inc., San Carlos, Ca) was performmed (Clement-Ziza, M., A. Munnich, et al. (2008). "Stabilization of RNA during laser capture microdissection by performing experiments under argon atmosphere or using ethanol as a solvent in staining solutions." RNA 14(12): 2698-704), with purification of amplified cDNA being achieved using QIAquick® PCR purification kit (Qiagen Ltd).
(Parameters: Extracted product = total_RNA, Amplification = OTHER: Ovation™ Pico WTA amplification system)
Laser capture microdissection (LCM): Serial horizontal cryosections were performed on paired lesional and non-lesional Lichen planopilaris (LPP) specimens (n=7 pairs). Slides containing bulge regions were identified as previously described using an adapted protocol (Ohyama, M., A. Terunuma, et al. (2006). "Characterization and isolation of stem cell-enriched human hair follicle bulge cells." J Clin Invest 116(1): 249-60). To preserve RNA integrity a rapid staining protocol was used (LCM Staining Kit, Ambion(R), Austin, Tx, USA) according to manufacturer’s instructions (Clement-Ziza, M., A. Munnich, et al. (2008). "Stabilization of RNA during laser capture microdissection by performing experiments under argon atmosphere or using ethanol as a solvent in staining solutions." RNA 14(12): 2698-704). Using a PALM(R) LCM microscope (Carl Zeiss Micro Imaging GmbH, Munich, Germany) a focused laser beam catapulted identified bulge epithelium into an RNase-free ependorff cap positioned above the section (x20 lens; power 90%; Suppl Fig A). Collected tissue was mixed with lysis buffer (Buffer RLT, RNeasy® Micro Kit, Qiagen® Ltd) before storage at -80°C.