Peripheral blood mononuclear cells (PBMC) were isolated using a Ficoll-Hypaque density gradient. After 24 hours of culture, non-adherent cells (mostly lymphocytes) were recovered, centrifuged, lysed and homogenized. Total RNA was isolated with the RNAeasy® Mini Kit extraction and purification system (QIAGEN Inc., Valencia, CA, USA) and analyzed for quality, integrity and DNA contamination by spectrophotometry and agarose gel electrophoresis prior to cDNA synthesis.
Label
Cy5
Label protocol
CodeLink™ Expression Bioarray System (GE Healthcare Biosciences Corp, Sunnyvale, CA, USA) was used for sample preparation and hybridization. Total RNA (0.5 μg) was primed for reverse transcription with oligo-d(T)24 containing the T7 RNA polymerase promoter site. After second-strand cDNA synthesis, the dscDNA served as the template for an in vitro transcription (IVT) reaction to produce the target cRNA. IVT was performed by the linear amplification method in the presence of biotin-11-UTP to label the target cRNA (Perkin Elmer, Boston, MA, USA). Labeling with Cy5-streptavidin was performed.
Hybridization protocol
After fragmentation, 8 µg of cRNA and CodeLink™ Human Whole Genome Bioarray were hybridized overnight at 37°C. The pre-arrayed 30-mer oligonucleotide slides contained over 45,000 well characterized human gene and transcripts.
Scan protocol
Slide was scanned at 635 nm for 3.5 s with 4.5 µm pixel size resolution in the arrayWoRx™ “e” scanner (Applied Precision, LLC, Issaquah, WA, USA.).
Description
The system first converts mRNA population in ds cDNA, followed by in vitro transcription and biotin labelling. Detection with Cy5-streptavidin.
Data processing
Image analysis and raw data were assessed with CodeLink™ Expression Analysis software v4.1. Values obtained were globally normalized to the median expression value of the whole array spots.
