Cells were transiently transfected with the indicated in vitro-synthesized siRNA (Invitrogen)
Growth protocol
HeLa cells were grown in Dulbecco’s Modified Eagle’s Medium (Hyclone) containing 10% fetal bovine serum (Hyclone).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Trizol (Invitrogen Life Technologies, Carlsbad, USA), purified using RNeasy columns (Qiagen, Valencia, USA) according to the manufacturers’ protocol. After processing with DNase digestion, clean-up procedures, RNA samples were quantified, aliquot and stored at -80°C until use. For quality control, RNA purity and integrity were evaluated by denaturing gel electrophoresis, OD 260/280 ratio, and analyzed on Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA).
Label
biotin
Label protocol
Total RNA was amplified and purified using the Ambion Illumina RNA amplification kit (Ambion, Austin, USA) to yield biotinylated cRNA according to the manufacturer’s instructions. Briefly, 550 ng 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, the cRNA was quantified using the ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA).
Hybridization protocol
750 ng of labeled cRNA samples were hybridized to each human-8 expression bead array for 16-18 h at 58°C, according to the manufacturer's instructions (Illumina, Inc., San Diego, USA).
Scan protocol
Detection of array signal was carried out using Amersham fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK) following the bead array manual. Arrays were scanned with an Illumina bead array Reader confocal scanner according to the manufacturer's instructions. Array data export processing and analysis was performed using Illumina GenomeStudio v2009.2 (Gene Expression Module v1.5.4).
Description
replicate 1 SAMPLE 3
Data processing
Array data export processing and analysis was performed usingIllumina GenomeStudio v2009.2 (Gene Expression Module v1.5.4). Array data were filtered by detection p-value < 0.05 (similar to signal to noise) in at least 50% samples (we applied a filtering criterion for data analysis; higher signal value was required to obtain a detection p-value < 0.05). Selected gene signal value was transformed by logarithm and normalized by quantile method. The comparative analysis between Control and Test samples was further carried out using fold-change.
