# Test file for GeneExpressionAssay (in vitro only)
# This assay is IN VITRO ONLY (supported_contexts: in_vitro_only)
# Performed on cell culture systems.
#
# Demonstrates a dose-response series as per-condition outputs: one
# AssayOutputMeasurement record per LPS concentration, each linked to its own
# exposure condition via measured_under.
gene_expression_assays:
  - id: "GE:001"
    name: "IL-8 gene expression across an LPS dose series"
    target_gene: "PR:000006689"
    has_exposure_condition:
      - id: "EXPOSURE:lps-vehicle-6h"
        name: "Saline vehicle for 6 hours (control)"
        exposure_duration:
          value: "6"
          unit:
            id: "UO:0000032"
            name: "hour"
      - id: "EXPOSURE:lps-10ng-6h"
        name: "LPS 10 ng/mL for 6 hours (low dose)"
        exposure_agent:
          id: "CHEBI:16412"
          name: "lipopolysaccharide"
        exposure_concentration:
          value: "10"
          unit:
            id: "UO:0000275"
            name: "nanogram per milliliter"
        exposure_duration:
          value: "6"
          unit:
            id: "UO:0000032"
            name: "hour"
      - id: "EXPOSURE:lps-100ng-6h"
        name: "LPS 100 ng/mL for 6 hours (high dose)"
        exposure_agent:
          id: "CHEBI:16412"
          name: "lipopolysaccharide"
        exposure_concentration:
          value: "100"
          unit:
            id: "UO:0000275"
            name: "nanogram per milliliter"
        exposure_duration:
          value: "6"
          unit:
            id: "UO:0000032"
            name: "hour"
    has_specified_output:
      - id: "GE:001-output-control"
        name: "Vehicle control IL-8 measurements"
        experimental_group: vehicle_control
        measured_under: "EXPOSURE:lps-vehicle-6h"
        mrna_level:
          value: "1.0"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 3
        protein_level:
          value: "1.0"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 3
        percentage_positive_cells:
          value: "12"
          unit:
            id: "UO:0000187"
            name: "percent"
          central_tendency: mean
          sample_size: 3
      - id: "GE:001-output-lps-low"
        name: "LPS 10 ng/mL IL-8 measurements"
        experimental_group: treated
        measured_under: "EXPOSURE:lps-10ng-6h"
        mrna_level:
          value: "1.9"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 3
          variability:
            variability_type: standard_deviation
            value: "0.3"
            unit:
              id: "STATO:0000169"
              name: "fold change"
        protein_level:
          value: "1.4"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 3
        percentage_positive_cells:
          value: "27"
          unit:
            id: "UO:0000187"
            name: "percent"
          central_tendency: mean
          sample_size: 3
      - id: "GE:001-output-lps-high"
        name: "LPS 100 ng/mL IL-8 measurements"
        experimental_group: treated
        measured_under: "EXPOSURE:lps-100ng-6h"
        mrna_level:
          value: "3.5"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 3
          variability:
            variability_type: standard_deviation
            value: "0.4"
            unit:
              id: "STATO:0000169"
              name: "fold change"
        protein_level:
          value: "2.1"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 3
        percentage_positive_cells:
          value: "45"
          unit:
            id: "UO:0000187"
            name: "percent"
          central_tendency: mean
          sample_size: 3
    gene_expression_method: "qRT-PCR"
    normalization_reference: "GAPDH"
    assay_date: "2024-10-01"
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-009"
      name: "Primary HBE ALI culture"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
      primary_cell:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      anatomical_origin:
        id: "UBERON:0002031"
        name: "bronchus"
      cell_culture_growth_mode: air_liquid_interface
      days_at_differentiation: 21
      donor_info: "Healthy donor, non-smoker, age 40"
      replicates_per_donor: 3
      substrate_type: transwell_insert
      passage_number: 2
    follows_protocols:
      - protocol_type: MolecularAssayProtocol
        id: "PROTOCOL:qpcr-002"
        name: "qRT-PCR for cytokine expression"
        detection_method: "TaqMan qPCR"
        reference_gene: "GAPDH"
        platform: "QuantStudio 6"

# Analysis layer: one comparison per dose, both against the same vehicle
# control output record.
response_comparisons:
  - id: "COMP:GE001-mrna-low"
    name: "Change in IL-8 mRNA, LPS 10 ng/mL vs vehicle"
    derived_from_assay: "GE:001"
    compared_measurement: "mrna_level"
    control_output: "GE:001-output-control"
    treated_output: "GE:001-output-lps-low"
    change_type: fold_change
    change_direction: increased
    change_value:
      value: "1.9"
      unit:
        id: "STATO:0000169"
        name: "fold change"
    p_value: 0.03
    statistical_test: "one-way ANOVA with Dunnett post hoc"
    derivation: reported_in_paper
  - id: "COMP:GE001-mrna-high"
    name: "Change in IL-8 mRNA, LPS 100 ng/mL vs vehicle"
    derived_from_assay: "GE:001"
    compared_measurement: "mrna_level"
    control_output: "GE:001-output-control"
    treated_output: "GE:001-output-lps-high"
    change_type: fold_change
    change_direction: increased
    change_value:
      value: "3.5"
      unit:
        id: "STATO:0000169"
        name: "fold change"
    p_value: 0.001
    statistical_test: "one-way ANOVA with Dunnett post hoc"
    derivation: reported_in_paper