Inguinal lymph node biopsies from 24 HIV-1-infected subjects at different clinical stages and 6 uninfected subjects were obtained for this University of Minnesota institutional review board–approved microarray study. Each lymph node biopsy was placed into a Falcon tube and snap-frozen by dropping it into liquid nitrogen. Frozen lymph nodes were homogenized with a power homogenizer (Heat Systems Ultrasonic) in TRIzol (In vitrogen, Cat# 15596-018) without thawing. Total RNA was isolated following the manufacturer’s protocol and further purified with an RNeasy Mini Kit (Qiagen, Cat# 74104).
Label
biotin
Label protocol
Double stranded cDNA and biotin-labeled cRNA probes were synthesized from 5 micrograms of total RNA with a MessageAmp II aRNA kit (Ambion, Cat# AM1757). The cRNA probes were column purified and fragmented with a Fragmentation kit (Ambion, cat# 8740).
Hybridization protocol
Fifteen micrograms of fragmented cRNA was hybridized to an Affymetrix Human Genome U133 Plus 2.0. After hybridization, chips were washed, stained with streptavidin-phycoerythrin, and scanned with GeneChip Operating Software at the Biomedical Genomics Center at the University of Minnesota. The experiments from each RNA sample were duplicated in the preparation of each cRNA probe and microarray hybridization. Microarray data analysis
Scan protocol
Affymetrix protocol
Description
none
Data processing
Cel. files were uploaded into the Expressionist program (Genedata, Pro version 4.5) and the expression level for each of the 47,000 transcripts in the arrays were analyzed by using the RMA algorithm. The expression levels from duplicated chips of the same individual’s RNA were correlated and averaged. The expression data from all individuals were exported as Excel files for statistical analysis. Tests for differences between the stages were conducted using the 2-sample t-test assuming the variance of the measurements are the same in the two groups. Fold differences in the level of gene expression between any 2 stages were estimated with the ratio of the means in the 2 stages. All computations were conducted using the software S-plus version 3.4 from MathSoft. After statistical analysis, data was sorted based on these transcript cutoffs: p-value of ≤ 0.05 and fold change ≥ 1.7. Significantly changed genes and transcripts were uploaded into NetAffix Analysis Center (http://www.affymetrix.com/analysis/index.affx) to query gene ontology information and into Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com) for gene annotation and pathway analysis.
