Quantigene viewrna assay kit

Cells were grown on poly-lysine coated chamber slides (BD Biosciences) additionally coated with gelatin. The QuantiGene ViewRNA assay kit (Affymetrix, Cat # QV0096) was used for detection of RNA hybridization signals. Cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with detergent solution for 5 min and then treated with protease for 10 min at room temperature. Each hybridization step, which was carried out in a hybridization oven at 40°C, was followed by three washes with wash buffer. Initially cells were incubated in an assay kit hybridization solution A containing a probe set for either the negative or positive stranded RNA rubella genome (complementary to the 5’ genomic region spanning nucleotides 1 to 6000) labeled with Cy5 (Affymetrix, custom made) in combination with a probe set for FITC-labeled housekeeping gene, peptidylpropyl isomerase B (PPIB, Affymetrix, Cat # VA4-10510), or a negative control probe set against Influenza A virus NP-negative strand labeled with Cy5 (Affymetrix, Cat# VF1-10583) in combination with a PPIB probe set. The cells were then incubated with hybridization preamplifiers and finally with labeled probes (1:100 in an assay kit hybridization buffer C), washed and imaged using a Zeiss fluorescent microscope. AxioVision software was used to create an outline of a cell periphery based on PPIB localization.