title: Optimizing high dimensional gene expression studies for immune response following smallpox vaccination using Taqman® low density immune arrays.
abstract: BACKGROUND
  We sought to determine the time and vaccinia virus dose combination that would maximize the number of acute immune response changes in response to vaccinia stimulation in preparation for a large gene expression microarray experiment.
  
  
  METHODS
  PBMCs from ten subjects were exposed to five vaccinia virus doses for three lengths of time. Gene expression was measured for 90 immune response genes via Taqman® Low Density Immune Arrays. Expression data were normalized via model-based non-linear normalization. Linear mixed effects model results were used to standardize changes across genes and determine the time/multiplicity of infection (MOI) combination with the largest number of changes.
  
  
  RESULTS
  The greatest number of changes occurred with a MOI of 5.0 and exposure time of 48 h. Further inspection revealed that most changes had occurred earlier and faded at this combination. The second highest number of changes was found at a MOI of 0.5 PFU/cell and time of 18 h.
  
  
  CONCLUSIONS
  We conclude a time of 18 h with a MOI of 0.5 PFU/cell is the optimal time/MOI combination for the full scale gene expression study. The strategy described herein is a general and resource efficient way to make critical decisions regarding experimental parameters for studies utilizing expensive assays that interrogate a large number of variables.
authors.affiliation: Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: Ann L
authors.initials: AL
authors.lastname: Oberg
authors.forename: Neelam
authors.initials: N
authors.lastname: Dhiman
authors.forename: Diane E
authors.initials: DE
authors.lastname: Grill
authors.forename: Jenna E
authors.initials: JE
authors.lastname: Ryan
authors.forename: Richard B
authors.initials: RB
authors.lastname: Kennedy
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Poland
mesh_terms: D000293:Adolescent; D000328:Adult; D057134:Antibodies, Neutralizing / Q000097:blood; D000914:Antibodies, Viral / Q000097:blood; D016207:Cytokines / Q000096:biosynthesis; D004306:Dose-Response Relationship, Immunologic; D020869:Gene Expression Profiling / Q000379:methods*; D005802:Genes, MHC Class II; D006801:Humans; D066298:In Vitro Techniques; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000821:virology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012900:Smallpox Vaccine / Q000008:administration & dosage* / Q000276:immunology; D014616:Vaccinia virus / Q000276:immunology; D055815:Young Adult
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D016207:Cytokines; D012900:Smallpox Vaccine