tissue: plucked anagen hair follicles gender: male age: 33 years old etenic hair group: african
Extracted molecule
total RNA
Extraction protocol
sample collection protocol: The collection procedure consisted first in the isolation of one hair with fingers protected by gloves and then pulling it close to the root with tweezers. After visually checking the integrity and the anagen stage, the follicle was immersed into a preservation solution (RNAlater, Ambion, Applied Biosystems, Foster City, Califórnia, EUA). Then, the excess of hair fiber was cut-off with scissors. The process was repeated until 50 follicles were collected from the entire scalp. The microtube was stored as soon as possible at 4°C and later transferred to -80°C. Samples were defrost on ice and the RNAlater was removed. Total RNA was extracted using the SV Total RNA Isolation system (Promega Corporation, Madison, Wisconsin USA) according to the protocol of the kit. RNA elution volume was 50 microliters/ 50 hair follicles sample. Concentration and purity was determined by spectrophotometry (NanoDrop ND-1000 UV/VIS, ThermoFisher Scientific,Waltham, Massachusetts, EUA) and integrity was confirmed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay (Agilent Technologies, Palo Alto, CA).
Label
biotin
Label protocol
Total RNA was processed for use on Affymetrix (Santa Clara, CA, USA) GeneChip HuGene 1.0 ST Arrays, according to the manufacturer’s Whole Transcript Sense Target Labeling Assay. Briefly, 100 ng of total RNA containing spiked in Poly-A RNA controls (GeneChip Expression GeneChip Eukaryotic Poly-A RNA Control Kit; Affymetrix) was used in a reverse transcription reaction (GeneChip® WT cDNA Synthesis Kit; Affymetrix) to generate first-strand cDNA. After second-strand synthesis, double-stranded cDNA was used in an in vitro transcription (IVT) reaction to generate cRNA (GeneChip® WT cDNA Amplification Kit; Affymetrix). 15 ug of this cRNA was used for a second cycle of first-strand cDNA synthesis (GeneChip® WT cDNA Synthesis Kit; Affymetrix). 5.5 ug of single stranded cDNA was fragmented and end-labeled (GeneChip® WT Terminal Labeling Kit; Affymetrix). Size distribution of the fragmented and end-labeled cDNA, respectively, was assessed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay.
Hybridization protocol
5 µg of end-labeled, fragmented cDNA was used in a 100-µl hybridization cocktail containing added hybridization controls. 80 µl of mixture was hybridized on arrays for 17 h at 45°C. Standard post hybridization wash and double-stain protocols (FS450_0007; GeneChip HWS kit, Affymetrix) were used on an Affymetrix GeneChip Fluidics Station 450.
Scan protocol
Arrays were scanned on an Affymetrix GeneChip scanner 3000 7G.
Description
Gene Expression from hair curvature classification: class V-VIII Black2P
Data processing
The 11 arrays were analyzed using Chipster 2.2 (Kallio et al., 2011) using custom cdf file HuGene10stv1_Hs_ENTREZG as available from Brainarray database version 14 (Sandberg & Larsson, 2007). Following RMA normalization and biomaRt annotation, differential expression was determined by empirical Bayes two-group test (Smyth, 2004) with Benjamini-Hochberg multiple testing correction and a p-value cut-off of 0.05.
