|Application:||Gene expression microarray analysis|
|Number of samples:||103|
|Release date:||Dec 8 2009|
|Last update date:||Dec 6 2012|
|Dataset link||Altered gene expression and DNA damage in peripheral blood cells from Friedreich's ataxia patients|
Peripheral blood samples were collected from 48 children with FRDA participating in a randomized, placebo-controlled clinical trial for idebenone. All whole blood samples in this study were collected before administration of idebenone. RNA was isolated utilizing the PAXgene™ blood RNA isolation kit (PreAnalytiX/ QIAGEN, Hilden, Germany) according to the manufacturer’s protocol, including the optional on-column DNase digestion, except that the centrifugation time after proteinase K digestion was increased from 3 to 20 minutes in order to obtain a tighter debris pellet. RNA quality was assessed with an Agilent Bioanalyzer™ (Palo Alto, CA) to ensure that samples with intact 18S and 28S ribosomal RNA peaks were used for microarray analysis. Twenty samples were lost during the isolation procedures with 28 high-quality RNA samples remaining. RNA was also isolated, using the same methods already described, from 14 FRDA adults and 15 healthy adults as controls. Lymphoblastoid FRDA cell line and age-matched control RNAs were isolated using the QIAGEN Rneasy midi (100–500 µg RNA) according to the manufacturer’s protocol. Gene expression profiling was conducted using Agilent Human 1A(V2) Oligo arrays with ~20,000 genes represented (Agilent Technologies, Palo Alto, CA). Each sample was hybridized against a human universal RNA control (Stratagene, La Jolla, CA). 500 ng of total RNA was amplified and labeled using the Agilent Low RNA Input Fluorescent Linear Amplification Kit, according to manufacturer’s protocol. For each two color comparison, 750 ng of each Cy3- (universal control) and Cy5-labeled (sample) cRNA were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 17 hours in a rotating hybridization oven according to the Agilent 60-mer oligo microarray processing protocol prior to washing and scanning with an Agilent Scanner (Agilent Technologies, Wilmington, DE). The data were obtained with the Agilent Feature Extraction software (v9.1), using defaults for all parameters. The Feature Extraction Software performs error modeling before data are loaded into a database system. Images and GEML files, including error and P values, were exported from the Agilent Feature Extraction software and deposited into Rosetta Resolver (version 5.0, build 18.104.22.168.48) (Rosetta Biosoftware, Kirkland, WA).
NIEHS Microarray Core
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