Comparative Analysis of Viral Gene Expression Programs during Poxvirus Infection: A Transcriptional Map of the Vaccinia and Monkeypox Genomes
Figure 1
Validation of poxvirus-human microarray.
(A) Monkeypox ORF PCR products were tested by printing the 384-well plate of monkeypox products on an array with three 384-well plates of human gene clones. Each spot from one well of a 384-well plate was printed 8 times (across, in rows). Two of the three 384-well plates of human clones were printed, then the monkeypox plate was printed (monkeypox spots start at the fifth row down, seventeenth spot from the left), and then the third 384-well plate of human clones was printed. Test hybridizations were performed using control uninfected cell line RNA from K562 cells (Uninfected Control RNA) or RNA from human PBMCs infected with monkeypox at an MOI of 1 at 24 hours post infection (Monkeypox RNA) labeled with Cy 5 versus common reference RNA (Stratagene, Inc.) labeled with Cy3. (B) 384-well plates of the ORF Monkeypox ORF PCR products were spotted in duplicate on the human array (bottom of the sector, Poxvirus spots). One of the 48 sectors is shown. Test hybridizations were performed with Cy5-labeled RNA from uninfected primary human monocytes or Cy5-labeled RNA from MPXV-infected primary human monocytes at 48 hours post-infection, versus a Cy 3 labeled common reference pool (Universal Human Reference, Stratagene Inc.) combined with an equal mix of poxvirus transcripts from all timepoints.