Streptavidin phycoerythrin

MGC-803 cells that have been transfected with si-CENPO and control MGC-803 cells were used to compare gene expression patterns by microarray analysis. Following transfection of MGC-803 cells with CENPO siRNA, total RNA was extracted and 50–500 ng of RNA was used to generate biotin-modified amplified RNA (aRNA) using a GeneChip 3′IVT Express kit (Affymetrix; Thermo Fisher Scientific, Inc.). Reverse transcription was performed using a T7 oligo (dT) primer and a first-strand IVT Labeling Master Mix was used to produce multiple copies of biotin-modified aRNA. The aRNA was then purified and quantified. Following fragmentation, the aRNA was hybridized to the GeneChip PrimeView™ human array (Affymetrix; Thermo Fisher Scientific, Inc.). The chips were subsequently stained with streptavidin-phycoerythrin (MoBiTec, Goettingen, Germany) for 10 min at 25°C and washed in a GeneChip Fluidics Station 450. The microarray signals were scanned and analyzed using the GeneChip Array Scanner 3000 (Thermo Fisher Scientific, Inc.). Differentially expressed genes (DEGs) between the two groups were defined as those exhibiting a >1.5-fold or a <0.5-fold-change in gene expression and where the P-value was <0.05 following correction. Ingenuity Pathway Analysis (IPA; http://www.ingenuity.com) (13 (link),14 (link)) was performed for significant DEGs and enrichment pathway analysis.

HCI-H1299 cells that have been transfected with shCENPO and shCtrl-HCI-H1299 cells were used to compare gene expression patterns by microarray analysis. Following transfection of HCI-H1299 cells with CENPO shRNA, total RNA was extracted used to generate biotin‑modified amplified RNA (aRNA) using a GeneChip 3′IVT Express kit (Affymetrix; Thermo Fisher Scientific, Inc.). Reverse transcription was performed using a T7 oligo (dT) primer and a first strand IVT labeling master mix was used to produce multiple copies of biotin‑modified aRNA. After fragmentation, the aRNA was hybridized with the GeneChip PrimeView™ human array (Affymetrix; Thermo Fisher Scientific, Inc.). The chips were then stained with streptavidin‑phycoerythrin (MoBiTec, Goettingen, Germany) for 10 min at 25 C and washed in a GeneChip Fluidics Station 450. The microarray signals were scanned and analyzed using the GeneChip Array Scanner 3000 (Thermo Fisher Scientific, Inc.). Differentially expressed genes (DEGs) between the two groups were defined as those exhibiting ∣logFC∣=1 in gene expression and where the P‑value was <0.05 following correction.