7 protocols
AccessionNameType
P-AFFY-6
feature_extraction
Title: Affymetrix CEL analysis. Description:
P-AFFY-13
hybridization
URL: http://www.affymetrix.com/support/technical/manual.affx
Affy_whole_transcript_labelling.pdf
This document describes Affymetrix recommended hybridization protocols for use with GeneChip Exon Arrays.
P-MTAB-31661
bioassay_data_transformation
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.
P-MTAB-31662
labeling
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)
P-MTAB-31663
grow
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)
P-MTAB-31659
nucleic_acid_extraction
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)
P-MTAB-31660
specified_biomaterial_action
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.