Genearray scanner g2500a

Independently derived 70Z/3 lines infected with MSCV, or MSCV-miR-24-2 were generated previously[65 (link)]. Four MSCV lines and 2 MSCV-miR-24-2 lines were examined. Total RNA was prepared using Trizol reagent (Invitrogen, Carlsbad, CA). RNA quality was evaluated using the Lab-on-a-Chip Bioanalyzer 2100 (Agilent, Palo Alto, CA). Biotinylated cRNA was prepared according to the standard Affymetrix protocol using the Superscript Choice System (Invitrogen) and the RNA transcript labeling kit (ENZO, Farmingdale, NY) for cRNA preparation. Following fragmentation 10ug of cRNA was hybridized to Affymetrix Mouse 430 2.0 Gene Chip. Microarray analysis was performed using the Affymetrix GeneArray Scanner G2500A. The Affymetrix Expression Console Software Version 1.4.0 was used to create summarized expression values (CHP-files) from expression array feature intensities (CEL-files). Raw data were normalized with robust multichip analysis (RMA) with Affymetrix transcriptome analysis console (TAC) software. DNA microarray and sample annotation data were deposited in GEO under the accession number GSE65874.

Total RNA was reverse transcribed to synthesis first-strand cDNA by using a T7-Oligao (dT) Promoter Primer, and then second-strand cDNA was synthesized and purified. Next, in in vitro transcription reaction, second-strand cDNA serves as a template performed for complementary RNA (cRNA) amplification and biotin labeling and was cleaned up and fragmented. All the operations were following the instructions of MessageAmp™ Premier RNA Amplification kit (Invitrogen, USA). Subsequently, the biotinylated cRNAs were hybridized to Affymetrix Human Genome U133A genechip overnight at 45°C with rocking. Genechip was then washed and stained according to the protocol of GeneChip® Hybridization, Wash, and Stain kit (Thermo Fisher Scientific, USA). Finally, Affymetrix Gene Array Scanner G2500A scanned the chip, and the scanned images were analyzed by MAS5.0 software.

After a 16-hour hybridization, the HG_U133A microarray was washed with buffer and stained with solutions (SAPE solution; streptavidin antibody solution) and then scanned with the GeneArray Scanner G2500A (Affymetrix).