Affymetrix human genechip u133 plus 2.0 array

In the current study, total B cells (purity > 97%) were isolated in PBMCs using B cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Total RNA from B cells was extracted using RNeasy Micro kit following the manufacturer’s protocol (Qiagen, Valencia, CA), quantified by Nanodrop 2000 (Nanodrop, Wilmington, DE), and qualified by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA concentration more than 20 ng/μL was selected to perform Affymetrix GeneChip assays, including high ANA HIV+ subjects (n = 5) and low ANA HIV+ subjects (n = 9) on D0 and D7 post-vaccination. Affymetrix Human GeneChip U133 Plus 2.0 Array was used for RNA hybridization and labeling assay according to the manufacturer’s protocol (Affymetrix). The scanned images and probe signals were analyzed by GCOS (Affymetrix). All microarray data and statistical analyses were performed using R (version 3.3.1). Normalization was performed with RMA algorithm which included background adjustment and quantile normalization [22 (link)]. Selection of distinct gene expression was identified by P value less than 0.05. Biological profiles of distinct genes were analyzed and clustered using iPathway (Advaita, Plymouth, MI). All microarray data are available in the GEO database.

PBMCs were prepared for gene expression analysis by removing the media from the cells, rinsing with room temperature phosphate buffered solution pH 7.4 (Quality Biologicals Inc., Gaithersburg, MD) and lysing the cells via resuspension in Buffer RLT (Qiagen, Valencia, CA). The lysed cell mixture was stored at −80 °C. Total RNA was isolated from PBMCs that underwent different storage conditions using Qiagen RNeasy kit by following manufacturer’s protocol. Total RNA was quantitated and qualified in Nanodrop 8000 and Agilent Bioanalyzer RNA Nano 6000 chip before performing Affymetrix GeneChip assays. Affymetrix Human GeneChip U133 Plus 2.0 Array (Affymetrix, Sacramento, CA) was used and labeling assay was performed by following manufacturer’s protocol.
Microarray data was normalized and analyzed using Partek Pro (St. Louis, MO). Genes were selected by 3-way ANOVA statistical analysis and correlated with cell viability flow cytometry data. The selection criteria are P-value less than 0.05 and absolute fold change more than 3. The correlation selection criteria are p-value is less than 0.05 and inter-quartile value is more than 1.4. Selected genes were normalized by medium shift before hierarchy clustering. Biological function annotation was done on in-house software DAVID [29 (link)] (http://david.abcc.ncifcrf.gov/) and Ingenuity IPA software (Qiagen, Germantown, MD).