Genechip 3 ivt plus kit

The microarray procedure was performed according to the Affymetrix protocols (Santa Clara, CA, USA). In brief, 100 ng of total RNA was amplified and labeled using the GeneChip® 3′ IVT Plus kit (Affymetrix, Santa Clara, CA, USA). Hybridization cocktails containing fragmented, end-labeled single-stranded cDNA were prepared and hybridized to the GeneChip Human Genome U133 Plus 2.0 Array for 16 h at 45°C. (Affymetrix, Santa Clara, CA, USA). The signal intensities were measured using a GeneChip Scanner 3000 7G (Affymetrix) and converted to numerical data using the GeneChip Command Console® Software (AGCC). The microarray data analysis was performed by Partek® Genomics Suite 6.6 (Partek Inc., St Louis, MO, USA) using the default Partek normalization parameters. Affymetrix CEL files were imported and background correction and normalization were performed using GC-robust multiarray average (GC-RMA) algorithm. Comparisons among treatment groups were performed with the ANOVA tool implemented in the Partek software after removal of batch effects. In order to correct for multiple comparisons and reduce the number of false positives, a false discovery rate (FDR) adjusted p value <0.05 was used in combination with a fold change (FC) ≥ 2. Biofunctional analysis was performed using ConsensusPathDB (http://cpdb.molgen.mpg.de) [20 (link)] and Partek Pathway software (Partek, St Louis, MO, USA).

Total RNA was extracted from sh-SKA1 and control 786-O cells and hybridized to the Gene Chip Primeview Human gene array (901838; Affymetrix) using the GeneChip^® 3′ IVT Plus Kit (Affymetrix) in accordance with the manufacturer’s instructions. Ingenuity Pathway Analysis (IPA) (Ingenuity^® Systems, https://www.ingenuity.com) was used to build networks and functional analysis. The top 535 genes, illustrating the most significant changes due to the knockdown of SKA1, were submitted to the above website. Graphs illustrating gene expression based on SKA1 silencing were generated using STATA (STATACorp 2015, College Station, TX, USA).

Total RNA was extracted from A549 cells infected with lentivirus expressing either shCPSF6 or shCtrl as described above. RNA integrity was assessed by NanoDrop 2000 (Thermo Fisher Scientific) and Agilent Bioanalyzer 2100 (Agilent Technologies Inc., Santa Clara, CA, USA). The RNA was amplified and Biotin-modified with GeneChip 3’IVT PLUS Kit (Affymetrix Inc., Santa Clara, CA, USA) to generate aRNA. The aRNA was fragmented and hybridized onto PrimeView Human Gene Expression Array (Affymetrix Inc.) according to the manufacturer’s guidance. GeneChip scanner 3000 (Affymetrix Inc.) was used to scan Microarray signals.
Differentially expressed genes (DEGs) were identified according to the following criteria: fold change > 2.0 and false discovery rate (FDR) < 0.05. The canonical pathways and associated protein networks involving in the identified DEGs were then established by Ingenuity Pathway Analysis (IPA) 9.0 (Ingenuity Systems, Redwood City, CA, USA).