BioProject

Human fibroblast monolayers plated at a density of 3 x 104 cells/cm2 in 850 cm2 roller bottles were infected at confluency (day 5 post-seeding) with purified virions of human cytomegalovirus (CMV) strain AD169varATCC (AD) or the AD169 US27/US28 deletion mutant virus RV101 (RV) at a multiplicity of infection of 10. More...

Human fibroblast monolayers plated at a density of 3 x 104 cells/cm2 in 850 cm2 roller bottles were infected at confluency (day 5 post-seeding) with purified virions of human cytomegalovirus (CMV) strain AD169varATCC (AD) or the AD169 US27/US28 deletion mutant virus RV101 (RV) at a multiplicity of infection of 10. After virus adsorption for 1h at 37C with 5% CO2, the inoculum was removed and the cells were washed twice with culture medium prior to the addition of fresh medium. At 50, 72 and 98 hpi, infected monolayers were harvested, snap-frozen and stored at -80:C until the time course was completed. Uninfected confluent HF cultures were used as reference. mRNA was extracted using the FastTrack. 2.0 mRNA Isolation Kit (Invitrogen, Carlsbad, CA). Only RNA preparations reaching OD260/280 ratio values of 2.0 were used. Labeled cDNA populations were synthesized from 2 microg of mRNA per sample by reverse transcription and simultaneous incorporation of Cy3- (reference sample, green) or Cy5- (infected samples, red) dUTP using an oligo-dT20 primer and the Superscript II Reverse Transcriptase. Reference and sample cDNA populations were mixed and applied on the arrays. Hybridizations were performed at 650C for 16 to 18 h in a custom slide chamber with humidity maintained by a small reservoir of 3x SSC. The arrays were then washed, scanned with a Gene Pix 4000A Scanner, gridded and flagged with Gene Pix Pro 4.0 software before submission to the SMD. Groups of assays that are related as part of a time series. Keywords: time_series_design Overall design: Computed Less...