Quantigene viewrna assay

WI-38 cells on poly-lysine coated chamber slides (BD Biosciences) were mock infected or infected with RV strains at MOI = 5 (two experiments with two replicate wells per each virus strain). RV genomic RNA was detected with the positive strand-specific probe set using the QuantiGene ViewRNA assay kit (Affymetrix, Cat # QV0096) as previously described [21 (link)].

Frozen blocks were sectioned at a thickness of 20 μm. Non-radioactive In situ hybridization was performed as described [14 (link)] using 1.2μg of either Wnt9b, Ret, or Wnt11 digoxigenin-labeled (DIG) riboprobes overnight at 68°C. Sections were treated with 2μg/ml RNase for 15 minutes at 37°C and incubated in anti-DIG-AP antibody (1:2000, Roche) at 4°C overnight and incubated with BM purple at room temperature to visualize signals. Sections were fixed in 4% PFA, mounted in glycergel mounting media (Vector, Burlingame, CA). In Situ Hybridization for Wnt4 was performed using the Affymetrix QuantiGene ViewRNA assay. Briefly, paraffin blocks were sectioned at a thickness of 5 μm, deparaffinized, boiled in pre-treatment solution (Affymetrix, Santa Clara, CA) and digested with proteinase K. Sections were incubated with a custom designed QuantiGene ViewRNA Wnt4 probe for 2 hrs at 40°C. Signal was amplified with Pre-Amp and Amp solutions and then developed Fast-Red Substrate. Slides were counterstained with DAPI, mounted with Fluoromount (Sigma, St. Louis, MO) and photographed on a Nikon 90i-eclipse upright microscope.