The methods for gene expression profiling were previously published (Joehanes and others 2013 ). Briefly, peripheral blood samples were extracted using the PAXgene Blood mRNA kit (PreAnalytiX, Hombrechtikon, Switzerland), and amplified by the WT-Ovation Pico RNA Amplification System (NuGEN, San Carlos, CA), according to manufacturer’s instructions. cDNA was then hybridized to the Human Exon 1.0 ST Array (Affymetrix, Inc., Santa Clara, CA) for quantification. The raw data were quantile-normalized and natural-log transformed, followed by summarization using Robust Multi-array Average (Irizarry and others 2003 (link)). The gene annotations were obtained from Affymetrix NetAffx Analysis Center (version 31). We excluded transcript clusters that were not mapped to RefSeq transcripts, resulting in 17,873 distinct transcripts (17,324 unique gene identifiers) for downstream analysis.
Netaffx analysis center version 31
Manufactured by Thermo Fisher Scientific
The NetAffx Analysis Center (version 3) is a web-based platform for gene expression data analysis. It provides researchers with tools for performing tasks such as gene annotation, pathway analysis, and differential gene expression analysis. The platform is designed to facilitate the interpretation and understanding of gene expression data generated from various experimental techniques.
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3 protocols using netaffx analysis center version 31
1
Profiling Gene Expression in Blood
2
Gene Expression Profiling from Blood Samples
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Gene expression profiling was performed from blood samples taken on Offspring examination 8 (n = 2442) and Gen 3 examination 2 (n = 3180) as previously described (Joehanes et al., 2013 ). In brief, the Affymetrix Human Exon 1.0 ST Array (Affymetrix, Inc, Santa Clara, CA) was used and gene annotations were obtained from Affymetrix NetAffx Analysis Center (version 31) resulting in 17,324 distinct genes for downstream analysis.
3
Framingham Gene Expression Profiling
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Framingham gene expression profiling has been described in detail48 (link)49 (link)50 (link). Briefly, total RNA was isolated from fasting peripheral whole blood collected during clinic visits. RNA was then amplified and reverse transcribed into cDNA, which was hybridized to the Human Exon 1.0st Array (Affymetrix, Santa Clara, CA) according to standardized protocols. We used Robust Multi-array Average51 method to normalize and summarize the raw data. The gene annotations were obtained from Affymetrix NetAffx Analysis Center (version 31). Only the most reliable probe sets derived from RefSeq and GenBank records were used in this study, corresponding to 17,873 distinct transcripts48 (link)49 (link)50 (link).
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