PMC:4979053 / 4795-6142 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4979053","sourcedb":"PMC","sourceid":"4979053","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4979053","text":"2.2. Sample Preparation and Hybridization\nThe cell lines were cultured at cell confluence according to the corresponding ATCC datasheets. Total RNA samples were extracted using the miRNeasy kit (Qiagen Spa, Hilden, Germany) and quantified by ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Using an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA), RNA integrity was assessed on the basis of the RIN (RNA integrity number) factor, and the presence of low molecular weight RNA (5S) was verified. Labeling of total RNA samples was performed using the FlashTagTM Biotin RNA labeling Kit (Genisphere Inc., Hatfield, PA, USA) starting from 1 μg of total RNA. Briefly, the tailing reaction is followed by ligation of the biotinylated signal molecule to the target RNA sample. The labeling reaction is based on Genisphere proprietary 3DNA dendrimer signal amplification technology. Prior to array hybridization, total RNA labeling was verified using the enzyme-linked oligosorbent assay (ELOSA). After that, biotin-labeled samples were hybridized onto the arrays at 48°C, then washed and stained using the Affymetrix Fluidics Station 450. Arrays were scanned with the GeneChip© Scanner 3000 7G to acquire fluorescent images of each array and analyzed by use of GeneChip Operating Software (GCOS, version 1.2).","divisions":[{"label":"Title","span":{"begin":0,"end":41}}],"tracks":[]}