Hs 400 hybridization station

cRNA sample preparation, labeling and hybridization were performed according to the manufacturer’s instructions. Briefly, 0,5 μg of total RNA was used for cDNA synthesis and amplification using the Message™Amp aRNA kit (ThermoFisher Scientific). Then 825 ng of cRNA labeled with Cy3/Cy5 dyes using the Arcturus® TURBO labeling™ Cy™3/Cy™5 Kit (Applied Biosystems, Netherlands) were hybridized to Human 4x44k Oligonucleotide Microarrays (Agilent Technologies, USA) using the HS 400 hybridization station (Tecan, Switzerland). Microarray slides were scanned using a LS Reloaded scanner (Tecan, Switzerland) for microarray image analysis, and the data generated were further analyzed using ImaGene ver. 9.0 (BioDiscovery, USA) and GeneSpring GX ver. 11.0 (Agilent Technologies, USA) software. Loess normalization was performed to adjust microarray data for variation. Gene expression fold change above 1.5 (with p-value < 0.05) was defined as differentially expressed between two conditions. KEGG pathway enrichment analysis was performed using Webgestalt online source [22 (link)]. Network construction analysis using the GeneMANIA plug-in of Cytoscape was performed to predict the most related genes of our gene sets [23 (link)]. All of the microarray data was deposited in a GEO Dataset database, Accession number GSE93228, (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE93228).

Total RNA was prepared from mammary tumors and from matched, adjacent, normal transgenic mammary glands. Exiqon microarrays (Qiagen Inc, Germantown, MD) were used to assess differential miRNA expression. Four independent sets of tumors and their matched, normal transgenic mammary glands were compared. Three sets were assayed in duplicate (dye swap) and one set was tested in only a single assay. The dye swap assay was not performed for this pair. Total RNA (1000 ng) was labeled using either Hy5 or Hy3 dye following the manufacturer’s protocol for the miRCURY LNA microRNA Array (Exiqon/Qiagen). The Hy5-labeled samples and Hy3-labeled samples were mixed pairwise and hybridized to the array. Hybridization and wash steps were performed using a Tecan HS400 Hybridization Station (Tecan, Austria). The miRCURY LNA array slides were scanned using GenePix 4200 (Molecular Devices, San Jose, CA). Differential miRNA expression was validated by reverse-transcriptase quantitative PCR (RT-qPCR). RT-qPCR was performed utilizing SYBR assays obtained from Exiqon (Qiagen Inc.), using the same four pairs of tumors and matched tissues that were used for the microarray experiments. These experiments also included two additional tumor-matched tissue pairs. Expression of potential downstream target genes was assessed using RT-qPCR.

cRNA sample preparation, labeling and hybridization was performed according to manufacturer’s instructions. Briefly, 1 μg of total RNA was used for cDNA synthesis and amplification using Message™Amp aRNA kit (ThermoFisher Scientific, USA). Then 825 ng of cRNA labeled with Cy3/Cy5 dyes using Arcturus® TURBO labeling™ Cy™3/Cy™5 Kit (ThermoFisher Scientific, USA) were hybridized to Agilent Mouse Whole Genome 4x44k Oligonucleotide Microarrays (Agilent Technologies, USA) using HS 400 hybridization station (Tecan, Switzerland). Three independent replicates of every sample were used. Microarray slides were scanned using LS Reloaded scanner (Tecan, Switzerland). Microarray image analysis and data generated were further analyzed using ImaGene ver. 9.0 (BioDiscovery, USA) and GeneSpring GX v11.5 software (Agilent Technologies, USA). Raw extracted gene expression data were normalized with Loess normalization to adjust microarray data for variation. Genes that showed expression values above fold change 1.5 (with p-value <0.05) were defined as differentially expressed in LLC1 cells between 2D and 3D cell culture conditions. Microarray design and data are available at the GEO database (Accession No. GSE75863 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE75863).