uveitis status: No uveitis gender: Female age: 32 set: 2 tissue: Blood disease state: Control
Treatment protocol
Whole blood was collected and incubated in 4 PAXGene tubes/subject (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) for two hours and stored frozen at -80C until the RNA was isolated.
Extracted molecule
total RNA
Extraction protocol
Total RNA isolation was performed using the PAXgene Blood RNA Isolation System (PreAnalytiX, a Qiagen BD Company, Valencia, CA, USA) following the manufacturer's recommendations. Following RNA isolation, DNase-treatment was performed as per PAXGene manufacturer’s recommendation. RNA recovery and quality was then assessed by examining UV 260/280 absorbance ratios and RNA size distribution on RNA Nano LabChips (Agilent Technologies, Santa Clara, CA, USA) processed on the Agilent 2100 Bioanalyzer. RNA from 4 PAXGene tubes was pooled for each subject.
Label
biotin
Label protocol
Affymetrix one-cycle cDNA synthesis/Affymetrix IVT with PNA treatment of total RNA: The standard Affymetrix amplification and labeling was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx (3 µg of total RNA as input), with the following modification: four peptide nucleic acid (PNA) oligonucleotides (Applied Biosystems, Foster City, CA, USA) designed to anneal to human α- and β-globin mRNA were added to the total RNA preparations immediately prior to initiation of the target synthesis step in order to bind globin mRNA and block cDNA synthesis. The PNA oligonucleotide stocks were prepared on day of use at concentrations recommended by Affymetrix (www.affymetrix.com/support/technical/byproduct.affx?product=bloodrna). Globin reduction PNA master mix was prepared by adding equal amounts of the globin PNA stocks. 5 µg of total RNA were incubated with oligo-dT primer and 1 µl PNA master mix for 10 minutes at 70C prior to first strand cDNA synthesis. Samples characterized as Set 1 were labeled and hybridized as one group and samples characterized as set 2 were processed as a second group a year later.
Hybridization protocol
Labeled cRNA targets were chemically fragmented at 95C for 35 min as per Affymetrix’s recommendations. The fragmented material was combined with Affymetrix hybridization controls: biotinylated control oligomer (B2) and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Following the manufacturer’s recommendations, 6.5 µg of target were hybridized with the Human Genome U133 Plus 2.0 GeneChip array (Affymetrix) for 16 hours at 45C. Post-hybridization array processing was performed on the Fluidics Station 450 (Affymetrix); array staining and signal amplification were performed according to manufacturer’s recommendations.
Scan protocol
The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan. The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software.
Description
n/a
Data processing
R2.4.0 64bit locally compiled by Sun compilers on Solaris 64/Bioconductor, gcrma background correction + invariant normalization + median polishing using PM only. Significance Analysis of Microarray (SAM) was utilized for statistical testing using samr package within R2.4.0 on Solaris 64. Sets 1 and 2 were normalized independently, and each normalization set included additional array data from subjects with other inflammatory disease diagnoses.
