The common reference cRNA sample comprised equal quantities of the following three components: (1) human umbilical cord endothelial cells (HUVEC), stimulated with TNF-alpha (50 microgram/ml, 6 hrs); (2) THP-1 cell line stimulated for 24 hours with phorbyl myristate (PMA, 100 microgram /ml), and subsequently for 6 hours with lipopolysaccharide (LPS, 1 microgram /ml), and (3) a mix of whole mount human aorta and iliac artery.
Biomaterial provider
Collaboration A.J.G. Horrevoets, M.J.A.P. Daemen, and T.J. van Berkel
Extracted molecule
polyA RNA
Extraction protocol
Total RNA of each one of the three reference components was isolated by Trizol, and treated with DNAse-I. Thereafter, mRNA of each component was linearly amplified for one round (MessageAmp aRNA kit, #1750, Ambion), synthesizing antisense cRNAs.
Label
Cy3
Label protocol
Within these cRNAs the molar ratio of incorporated aminoallyl-rUTP (A5660, Sigma, St. Louis, MO) to rUTP was 1:1. Cy3 mono-reactive dyes (PA23001, Amersham Biosciences, Piscataway, NJ) were coupled according to the manufacturer’s instructions. Labeled cRNA was purified using the RNeasy purification kit (Qiagen GmbH, Hilden, Germany).
Macrophages were isolated from human carotid artery plaque by Laser Capture Microdissection (Arcturus LCM system). After isolation of total RNA molecules, the samples were linearly amplified (MessageAmp aRNA kit, #1750, Ambion), synthesizing antisense cRNAs.
Label
Cy5
Label protocol
Within these cRNAs the molar ratio of incorporated aminoallyl-rUTP (A5660, Sigma, St. Louis, MO) to rUTP was 1:1. Cy5 mono-reactive dyes (PA25001, Amersham Biosciences, Piscataway, NJ) were coupled according to the manufacturer’s instructions. Labeled cRNA was purified using the RNeasy purification kit (Qiagen GmbH, Hilden, Germany).
Hybridization protocol
Each cRNA sample (1 micrograms; Cy5-labeled) was hybridized in duplicate against a common reference cRNA sample (1 micrograms; Cy3-labeled), for 16 hours at 40 degrees Celsius.
Scan protocol
Agilent scanner: resolution: 10 micrometer, scan area: 60 x 21.6 mm, PMT: 50%, Laser power: 50%
Description
See the methods section of the linked publication for additional information.
Data processing
After print-tip Loess-normalization (Loess span: 0.3; limma package, Bioconductor, available online: http://www.bioconductor.org), and background subtraction, the microarray intensity data imported in the Rosetta Resolver database.
