The following procedures were carried out by Macrogen Co. (Seoul, Korea). Five hundred fifty nanograms of total RNA was reverse-transcribed to cDNA using a T7 oligo(dT) primer. Second-strand cDNA was synthesized, in vitro transcribed, and labeled with biotin-NTP. After purification, 750 ng of labeled cRNA was hybridized to Illumina Human HT12 v.4 bead array (Illumina, San Diego, CA, USA) for 16-18 h at 58oC. The array signal was detected by using Amersham fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK). Arrays were scanned with an Illumina bead array Reader confocal scanner. Array data were filtered by detection p-value < 0.05 (similar to signal to noise). The average signal values of filtered genes were transformed by logarithm and normalized by the quantile method [8 (link)].
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Illumina Microarray Gene Expression Analysis
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Corresponding Organization :
Other organizations : Kangwon National University, Seoul National University
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Variable analysis
independent variables
- RNA concentration (550 ng of total RNA)
- Gene expression levels
- T7 oligo(dT) primer for reverse transcription
- Illumina Human HT12 v.4 bead array platform
- Hybridization conditions (16-18 h at 58°C)
- Amersham fluorolink streptavidin-Cy3 for signal detection
- Illumina bead array Reader confocal scanner for array scanning
- Detection p-value < 0.05 for data filtering
- Logarithm transformation and quantile normalization of gene expression data
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