Individual hairs were grasped as near to the scalp as possible and then yanked quickly out. The bottom centimeter of each hair, containing the hair follicle, was trimmed off into a 5ml specimen jar filled to the top with RNALater (Ambion, Austin, Texas). Samples were stored at ambient temperature until reaching the analytical laboratory, a period of 1-4 hr.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted on the day of collection or following storage at 4oC for 1-9 days in RNAlater. The hair follicle samples from each individual were removed from RNALater and, without verifying their stage, placed directly into a 2 ml microcentrifuge tube containing 1 ml Trizol™. RNA extraction was then conducted according to manufacturer’s instructions (Invitrogen Life Technologies, CA); a handheld homogenizer (Fisher Scientific, Hampton NH) was used to break up the samples. At the isopropanol precipitation step, RNA was pelleted either immediately or following storage at -80oC for up to seven months. After centrifugation, the pellet was washed with 70% ethanol, air dried and resuspended in 10 µl RNA free water (Gibco-BRL, Gaithersburg, MD). Concentration of the eluted RNA was quantified using GeneQuant spectrophotometer (Pharmacia Biotech, now Amersham Pharmacia Biotech, Piscataway, NJ) by absorbance readings at 260 and 280nm.
Label
biotin: double amplication using MessageAmp aRNA Kit (Ambion)
Label protocol
Briefly, first and second strand cDNA were synthesized. Unlabelled aRNA was generated by in vitro transcription with unbiotinylated NTPs. For probe preparation, aRNA was reverse transcribed with second round primers. Second-strand cDNA was synthesized with T7 Oligo(dT) primer and purified. Biotin-labeled cRNA was generated by in vitro synthesis transcription, and purified with Qiagen RNeasy kit.
Hybridization protocol
Each labeled cRNA was then fragmented, added to a hybridization solution, and hybridized for 16 hours to a HG-Focus genechip (Affymetrix, Santa Clara, CA) in an Affymetrix Fluidics Station 400.
Scan protocol
The chips were washed, stained with phycoerythrin-conjugated streptavidin and amplified by biotinylated anti-streptavidin. After a final wash the arrays were scanned in a GCS3000 (Affymetrix, Santa Clara, CA).
