Animal
Agriculture in the
New Biological
Economy
•Very high conservation amongst
vertebrates for all biological domains
•Anatomy
•Physiology
•Genomics
•Gene Expression
•Protein Function
•Regulatory and Signaling Pathways
•etc.
•The Amount of data being generated
surpasses our ability to capture its value.
•Future Biology will rely on artificial
intelligence to capture and analyze the
data.
•Animal Agriculture must evolve to leverage
new technology.
What types of data do we want to
remember and analyze?






LIMS Data – Historical information about laboratory data
generation and analysis
Phenotypic Data – The observable traits or characteristics of
an organism
Expression Profiling Data – What genes are expressed
when and where in an organism
Genetic Mapping Data – The relative positions of genes on
a chromosome and the distance between them.
Physical Mapping Data – A chromosome map of a species
that shows the specific physical locations of its genes and/or
markers on each chromosome.
Gene and Protein Functional Data – What does it do and
how?
Molecular
Genetics
Classical
Genetics
Comparative
Genomics
Phenotype
Sequence
Functional
Genetics
Genomics
Genotype
Proteomics
Minnesota Animal Genome
and Ontology Database
Sequencer
Pipeline
Markers
Clones
Genotype
Animal
Biological
Sample
Libraries
Phenotype
Arrays
Phenotype
Ontology
Annotation
Sequence
Sequence
Analysis
Oligos
oligo_in_project
oligo_id (FK)
project_id (FK)
oligo_sent_for_rh
rh_panel
sent_to
oligo_id (FK)
date_sent
sent_by
oligo_rh_volume_sent
oligo_rh_concentration_sent
oligo_submission
oligo_id (FK)
person_id (FK)
date
project_id (FK)
genotype
animalid (FK)
seq_mask_run_parameter
mask_run_parameter_id
maskid (FK)
masking_group_id (FK)
mask_run_parameter_name
mask_run_parameter_value
sqs_version_id (FK)
snp_quality
seq_orientation
conflict_flag
seq_reaction
source_dna_id (FK)
genotype_set
animalid (FK)
project_id (FK)
seq_snp
seq_snp_quality
haplotyping_method
program_id
version
algorithm
author
program_name
update_date
dye_chemistry
sequence_plate_id (FK)
sequencing_oligo_run
seq_rxn_address_row
seq_rxn_address_column
genotype_haplotype
haplotype_id (FK)
animalid (FK)
software_version
sqs_version_id
software_name
software_version
install_date
uninstall_date
sqs_notes
probability
seq_quality_run_parameter
sequence_quality_run_id (FK)
parameter_id
parameter_name
parameter_value
haplotype_run
haplotype_id
haplotype_string
frequency
parent
program_id (FK)
seq_plate
sequence_plate_id
plate_name
plate_format
plate_run_number
plate_application
cd_name
seq_plate_originator
plate_alias
seq_extraction
seq_extraction_run_id
seq_extraction_time
seq_extraction_method
number (FK)
seq_working_set_in_project
workingset_groupid (FK)
user_id (FK)
project_id (FK)
submitted_trace
submission_id (FK)
workingset_groupid (FK)
far_id (FK)
user_id (FK)
chromat_submit_format
chromat_submit_date
chromat_format_method
chromat_accession_number
chromat_file_path
chromat_run_time
submitted_seq
seq_id (FK)
seq_submitted_name
submission_id (FK)
workingset_groupid (FK)
phred_run_chromat
phd_file_id (FK)
sequence_quality_run_id (FK)
far_id (FK)
location
location_id
location_description
state
city
country
person
person_id
last_name
first_name
initials
isauthor
isinvestigator
email
login
person_status
person_telephone
mixed_sex_population
mixed_sex_population_id
breed_composition_id (FK)
species_id (FK)
marker
marker_id
marker_type
marker_name
marker_curator
public_marker
international_number
marker_type_description
object_id (FK)
db_id (FK)
breed_composition
breed_composition_id
species_id (FK)
breed_composition_description
rflp
marker_id (FK)
probe
animal_to_insertion
animalid (FK)
insertion_id (FK)
person_id (FK)
end
length
status
bac_assignment
bac_assignment_id
marker_id (FK)
chromosome_id (FK)
species_id (FK)
information_control
sequencing_reaction_in_far
far_id (FK)
source_dna_id (FK)
insertion
insertion_id
person_id (FK)
external_id
db_modification_date
marker_sequence
species_id (FK)
marker_id (FK)
somatic_cell_hybrid_result
sch_result_id
marker_marker (FK)
chomosome_assignment_marker (FK)
sex_segregated_population
sex_segregated_population_id
breed_composition_id (FK)
species_id (FK)
marker_reference
reference_id (FK)
marker_id (FK)
map_position
marker_id (FK)
map_id (FK)
linkage_group_id (FK)
map_position_order
map_position_position
map_position_linkage_group
map
map_id
freeze_date
mapping_method
map_curator
map_date
map_program
map_program_version
contribution_to_ref_by_author
reference_id (FK)
person_id (FK)
investigator_of_project
person_id (FK)
project_id (FK)
tissue_type
tissue_type_id
tissue_type_name
animal_in_annotation
animalid (FK)
forward_oligo_id (FK)
reverse_oligo_id (FK)
genomic_library
library_id (FK)
tissue_type_id (FK)
genomic_library_name
species_id (FK)
tissue
tissue_id
animalid (FK)
tissue_collection_date
tissue_quantity
tissue_tower
tissue_box
tissue_row_of_box
tissue_column_of_box
tissue_type_id (FK)
location_id (FK)
map_reference
reference_id (FK)
map_id (FK)
chromosome_assignment_reference
marker_id (FK)
reference_id (FK)
chromosome_id (FK)
species_id (FK)
tissue_in_tissue_sample
tissue_id (FK)
animalid (FK)
tissue_sample_id (FK)
lab
lab_id
lab_name
lab_institution
project_animal_group
animalid (FK)
project_id (FK)
marker_status
animalid (FK)
species_id (FK)
lab_id (FK)
reference
reference_id
year
reference_volume
beginning_page
ending_page
journal
title
cdna_plate
cdna_plate_id
source_dns_plate_id (FK)
animal
animalid
species_id (FK)
external id
animal_date_of_birth
animal_date_of_death
adminal_db_modification_date
breed (FK)
breed_species (FK)
linkage_group_map_pair
map_id (FK)
linkage_group_id (FK)
log_likelihood
species
species_id
species_name
cdna_library
library_id (FK)
tissue_type_id (FK)
cdna_library_name
species_id (FK)
assembly_parameter
assembly_parameter_id
assembly_parameter_value
assembly_parameter_name
sqs_version_id (FK)
size
marker_id (FK)
pcr_product_as_clone_source
library_id (FK)
old_source_dna_id (FK)
pcr_rxn_id (FK)
forward_oligo_id (FK)
peptide_fasta
seq_ohr
contig
contig_id
singleton_type
assembly_run_id (FK)
singleton_desc
contig_name
consensus_fasta
consensus_seq_length
contig_member_count
current_contig_datafile
poly_directory
contig_quality_method
contig_consensus_quality
contig_source
member_in_contig
contig_id (FK)
seq_id (FK)
contig_position
contig_id (FK)
chromosome_id (FK)
assembly_run_id (FK)
chromosome_start
chromosome_end
pcr_product_annotation
project_id (FK)
oligo_pair (FK)
library_relationship
cloning_source (FK)
cloning_result (FK)
seq_peptide
seq_id (FK)
start_ohr
singleton
assembly_run_id (FK)
source_submission
source_dna_submission_id
submitter_id (FK)
date
format
sample_number
project_id (FK)
submission_type
contig_in_annotation
project_id (FK)
contig_id (FK)
oligo_pair (FK)
assembly_run_id (FK)
sequence
seq_genbank_accession
seq_description
seq_name
seq_oligo_seq
seq_raw_fasta
seq_type
seq_length
seq_plate_id (FK)
seq_access_control_code (FK)
seq_run_number
seq_submission_status
seq_quality_score
seq_masked_fasta
seq_curator
seq_processing_method
seq_genbank_index
seq_file_path
seq_quality_run_id (FK)
effective_end_position_member
effective_start_position_member
member_orientation
member_start_pos_in_contig
pcr_product_from_rxn
source_dna_id (FK)
pcr_rxn_id (FK)
forward_oligo_id (FK)
source_dna_by_submission
source_dna_id (FK)
source_dna_submission_id (FK)
number_of_product
library
library_id
library_type
library_name
vector
titer
average_insertion_length
insertion_for
host
who_made_it
storage_location
number_of_clones
library_file
library_is_arrayed animal_source_in_source_dna
library_number_of_plates
source_dna_id (FK)
animalid (FK)
seq_workingset_member
workingset_groupid (FK)
user_id (FK)
seq
sequenom_oligo
oligo_number
oligo_name
contig_member_feature
contig_id (FK)
seq_id (FK)
contig_feature_id (FK)
member_feature_type
member_feature_name
member_feature_start_position
member_feature_end_position
sequenom_oligo_in_seq
seq_id (FK)
end_of_oligo
begin_of_oligo
end_of_seq
begin_of_seq
oligo_seq
contig_feature
contig_id (FK)
contig_feature_id
assembly_run_id (FK)
iub_code (FK)
contig_feature_start_position
contig_feature_end_position
contig_feature_type
contig_feaure_name
clone
clone_id
clone_plate_id (FK)
clone_address_row
clone_previous_plate_id
clone_previous_address_row
clone_address_column
clone_prev_address_column
clone_external_id
clone_alias
polymorphism_definition
iub_code
desc
nucleotide
pcr_product_as_source_dna
current_source_dna_id (FK)
old_source_dna_id (FK)
pcr_rxn_id (FK)
forward_oligo_id (FK)
blast_result
blast_id (FK)
blast_file
is_null
blast_software
blast_software_id
blast_program_name
blast_program_version
blast_run
blast_id
blast_software_id (FK)
blast_time
blast_type
db_id (FK)
source_dna
source_dna_id
source_dna_type
project
project_id
project_description
project_name
investigator_id (FK)
linkage_group
linkage_group_id
chromosome_id (FK)
assembly_run
assembly_run_id
assembly_method
elapsed_time
sqs_version_id (FK)
assembly_parameter_id (FK)
assembly_parameter_name (FK)
assembly_parameter_value (FK)
nearest_gene
other_id
nearest_gene_description
relative_position
distance
cm_position
sm_strand
chromosome_id (FK)
oligo_pair_penalty
compl_any
compl_end
product_size
capillary_run
far_id (FK)
chromat_directory
chromat_name
well_address
pcr_product
forward_oligo_id (FK)
pcr_product_in_project
project_id (FK)
oligo_pair
last_modify_time
embryo_collection
embryo_collection_id
sire_of_population (FK)
dam_of_population (FK)
breed_composition_id (FK)
species_id (FK)
be_sired_by_animal (FK)
be_dammed_by_animal (FK)
oligo_pair
forward_oligo_id (FK)
far
far_id
machine_id (FK)
array_use_count
far_date
far_operator
polymer_type
polymer_lot
polymer_install_date
array_serial_number
array_install_date
buffer
far_start_time
far_end_time
far_run_start_date
run_id
nearest_gene
mapping_id (FK)
annotation_project_id (FK)
insertion_id (FK)
genome_version_id (FK)
nuc_position (FK)
person_id (FK)
sequence_mapping
mapping_id
nuc_position
insertion_id (FK)
genome_version_id (FK)
person_id (FK)
person_in_project
group_member_id (FK)
project_id (FK)
investigator_id (FK)
join_date
obj_db
object_id
db_id (FK)
description
obj_type
chromosome
chromosome_id
species_id (FK)
chromosome_name
chromosome_length
pcr_product_in_far
far_id (FK)
source_dna_id (FK)
pcr_rxn_id (FK)
forward_oligo_id (FK)
annotation_project
annotation_project_id
proejct_name
ap_version
release_date
star
marker_id (FK)
two_point_linkage
map_id (FK)
used_as_new_marker (FK)
used_as_map_marker (FK)
member_in_masking_seq_group
masking_group_id (FK)
machine_id
machine_type
machine_name
genome_version
genome_version_id
genome_version_source
genome_version_description
genome_version_release_date
control_descripition
enzyme
enzyme_id
marker_id (FK)
chromosome_assignment
marker_id (FK)
chromosome_id (FK)
species_id (FK)
submission
submission_id
workingset_groupid (FK)
user_id (FK)
person_id (FK)
submission_date
submission_type
library_file
contact_file
publication_file
terminal_length
terminal_quality
internal_mask_length
intenal_mask_quality
internal_cont_length
internal_cont_quality
project_id (FK)
mask_seq_group
masking_group_id
fa
insertion_sequence
insertion_id (FK)
person_id (FK)
rapd
marker_id (FK)
external_db
db_id
db_name
release
db_version
oligo_strand
oligo_strand_id
oligo_id (FK)
oligo_strand
oligo_as_source_dna
source_dna_id (FK)
oligo_id (FK)
check_marker_phenotype
location_id (FK)
animalid (FK)
species_id (FK)
marker_id (FK)
lab_id (FK)
sscp
marker_id (FK)
gel_type
gel_temperature
gel_time
gel_length
gel_width
gel_thickness
voltage
amperage
wattage
quality_status
isnew
trim_fasta
trim_quality
seq_file
trim_start_position
trim_end_position
trim_fasta_length
mask_fasta_file
mask_fasta_file_id
file_name
file_path
genomic_dna_as_source_dna
genomic_dna_id (FK)
animalid (FK)
source_dna_id (FK)
plate_id (FK)
rna_prep
cdna_prep
cdna_id
cdna_preparation_type
cdna_preparation_date
cdna_preparation_person
reverse_transcription_oligo
reverse_transcription_condition genomic_dna
second_strand_oligo
second_strand_synthesis_positions genomic_dna_id
animalid (FK)
plate_id (FK)
tissue_sample_id (FK)
genomic_dna_row
genomic_dna_column
genomic_dna_preparation_date
genomic_dna_preparation_type
genomic_dna_preparer
tissue_sample
tissue_sample_id
tissue_sample_description
clone_as_source_dna
clone_id (FK)
clone_plate_id (FK)
library_id (FK)
source_dna_plate
source_dns_plate_id
source_dna_plate_format
source_dna_plate_name
pcr_rxn_in_plate
pcr_plate_id (FK)
source_dna_id (FK)
pcr_rxn_id (FK)
rna_prep_type
rna_prep_date
rna_prep_person
rna_prep_yield
rna_prep_id
tissue_sample_id (FK)
cnda_in_plate
cdna_id (FK)
rna_to_cdna
source_dna_id (FK)
cdna_id (FK)
cdna_plate_id (FK)
cdna_plate_row
cdna_column
cdna_prep_as_source_dna
cdna_id (FK)
project_species_element
species_id (FK)
project_id (FK)
pcr_reaction_score
pcr_reaction_score
pcr_reaction_desc
pcr_reaction
pcr_rxn_id
source_dna_id (FK)
forward_oligo_id (FK)
pcr_plate
pcr_plate_id
pcr_plate_name
source_dns_plate_id (FK)
pcr_reaction_date
pcr_reaction_person
pcr_reaction_machine
pcr_condition_id (FK)
pcr_condition
pcr_condition_id
dna_concentration
magnesium_concentration
dntp_concentration
polymerase_type
polymerase_amount hyb_probe_in_experiment
hybridization_probe_id (FK)
pcr_volume
hybridization_experiment_id (FK)
pcr_profile
source_dna_id (FK)
pcr_annealing_temp
pcr_profile_id (FK)
source_dna_as_hyb_target
source_dna_id (FK)
hyb_target_description
hyb_target_in_experiment
hybridization_experiment_id (FK)
source_dna_id (FK)
target_quantity_in_moles
genomic_dna_plate
plate_id
source_dns_plate_id (FK)
clone_plate
clone_plate_id
library_id (FK)
clone_plate_format
clone_plate_name
clone_plate_creation_date
clone_plate_storage_location
thaw_count
last_thaw_date
replication_date
replication_plate_id
replication_storage_location
source_dns_plate_id (FK)
pcr_rxn_as_source_dna
pcr_rxn_id (FK)
source_dna_id (FK)
forward_oligo_id (FK)
source_dna_in_plate
source_dna_plate_id (FK)
source_dna_id (FK)
source_dna_address_row
source_dna_address_column
blast_run_member
blast_id (FK)
seq_id (FK)
contig_id (FK)
assembly_run_id (FK)
oligo_id (FK)
pcr_additional_oligo_group
additioanl_oligo_set_id
pcr_profile
pcr_profile_id
pcr_step
pcr_instruction
source_dna_as hyb_probe
hybridization_probe_id
source_dna_id (FK)
specific_activity
probe_concentration
hyb_target_on_subarray
source_dna_id (FK)
subarray_id (FK)
array_id (FK)
subarray_column_address
subarray_row_address
hyb_experiment
hybridization_experiment_id
hyb_experiment_condition
hyb_experiment_stringency
hyb_experiment_type
hybridization_date
marker_within_source_dna
marker_id (FK)
source_dna_id (FK)
blast_address
blast_id (FK)
member_id (FK)
begin_of_subject
end_of_subject
begin_of_query
end_of_query
object_id (FK)
score
expected_value
identity
positive
hyb_subarray
subarray_id
array_id (FK)
total_subarray_row
total_subarray_column
array_row_address
array_column_address
blast_translation
blast_id (FK)
hyb_array
array_id
array_name
total_array_row
total_array_column
number_of_element_on_array
array_printing_date
array_printer_type
array_printing_person
array_slide_manufactuer
slide_coating_method
slide_coating_date
array_design_date
array_owner
oligo_purchase
purchased_date
oligo_id (FK)
purchased_by
purchased_from
purpose
oligo_design_parameter
oligo_design_parameter_id
oligo_design_run_id (FK)
oligo_design_parameter_name
oligo_design_parameter_description
seq_working_set
workingset_groupid
user_id (FK)
workingset_groupname
workingset_creationdate
group_annotation
pi_id (FK)
population_as_parent
population_as_parent_id
mixed_sex_population_id (FK)
breed_composition_id (FK)
species_id (FK)
microsatellite
marker_id (FK)
repeat_composition
oligo_name
oligo_curator (FK)
oligo_id (FK)
oligo_name
oligo_desc
oligo
oligo_id
oligo_seq
oligo_type
penalty
tm
gc_percent
self_any
self_end
end_stability
extinction_coeffecient
oligo_design_run_id (FK)
binding_site
oligo_design_run
oligo_design_run_id
oligo_design_run_date
sqs_version_id (FK)
sequence_mask_run
maskid
masking_group_id (FK)
masking_method
masking_time
mask_fasta_file_id (FK)
sqs_version_id (FK)
workingset_groupid (FK)
user_id (FK)
quality_file
phd_file_id
sequence_quality_run_id (FK)
far_id (FK)
phd_file_name
phd_file_path
phd_file_size
sequence_quality_run
sequence_quality_run_id
chromat_file
call_method
sequence_quality_run_time
sqs_version_id (FK)
sqr_raw_fasta_file_path
sqr_raw_fasta_file_name
far_id (FK)
oligo_curator
oligo_curator
public_oligo
oligo_storage
oligo_storage_id
oligo_id (FK)
plate
plate_address
box
box_address
member_in_pcr_additional_oligo_group
additional_oligo_set_id (FK)
oligo_id (FK)
oligo_orientation
oligo_forward_in_sequence
seq_id (FK)
oligo_position
mispriming_score
oligo_reverse_in_sequence
seq_id (FK)
oligo_position
mispriming_score
A relational data model for gene
expression analysis
 Track
microarray production and use
 Track tissues and probe generation
 Track hybridizations and data acquisition
 Track data normalization and submission
 Track analysis
 Interpretation
Statistical Analysis
•
•
•
•
Model: Y =X + a + b +
F-test
T-test
Fold Change
• Slide Quality
• Data Quality
• Results
• Spot Diameter
• Spot Area
• Footprint
• Front Channel and Background
Channel Signal Uniformity
• Dynamic Range
• Signal-Noise Ratio
• Signal Distribution of
Front/Background Channel
• Sample Correlation
and Cluster
• Differential expressed
…
genes
• Sample and gene expression
pattern cluster
• Gene/pathways identification
Animal
Species
Clone
Plate
Tissue
Genomic
DNA
Prep
Library
Clone
Source
DNA
RNA
Prep
cDNA
Prep
PCR
RXN
PCR
Product
Tissue
in
Tissue
Sample
Tissue
Sample
Primer
Pair
Animal to Source DNA
F
R
Primer
oligo_as_source_dna
source_dna_id (FK)
oligo_id (FK)
Remembering the Hybridization
source_dna
source_dna_id
source_dna_type
hyb_probe_in_experiment
hybridization_probe_id (FK)
hybridization_experiment_id (FK)
source_dna_id (FK)
source_dna_as_hyb_target
source_dna_id (FK)
hyb_target_description
hyb_target_in_experiment
hybridization_experiment_id (FK)
source_dna_id (FK)
target_quantity_in_moles
source_dna_as hyb_probe
hybridization_probe_id
source_dna_id (FK)
specific_activity
hyb_array
probe_concentration
array_id
array_name
total_array_row
total_array_column
hyb_target_on_subarray
source_dna_id (FK)
subarray_id (FK)
array_id (FK)
subarray_column_address
subarray_row_address
hyb_experiment
hybridization_experiment_id
hyb_experiment_condition
hyb_experiment_stringency
hyb_experiment_type
hybridization_date
hyb_subarray
subarray_id
array_id (FK)
total_subarray_row
total_subarray_column
array_row_address
array_column_address
array_printing_date
array_printer_type
array_printing_person
array_slide_manufactuer
slide_coating_method
slide_coating_date
array_design_date
array_owner
Porcine Microarray Platform
Pig Array-Ready Oligo Set v.1.0
13,297 70-mer oligonucleotide microarray.
Chip Platform:
•Slide: Corning GAPS II (25 X 75 X 1.1 mm)
•Print Conditions: 3X SSC;
•Oligo concentration printed: 20 μM
•Print temperature: Room temp (72-74 F);
•Print pin number: 48;
•Spot diameter: 140-160 μm;
•Distance between spots: 240 μm
Blastn
13,297 oligos
perfect match
Blastn
Ensembl DNA
TIGR TC hits
Blastn
*
Ensembl cDNA
The top hit containing a bit score >
100, an E-value < 0.001, and
similarity > 50%
*
Blastx
Ensembl peptide
Gene Ontology
*
Database
Start
Sequence
Number
Total Hit *
Unique Hit
Duplicates
No
Hit*
Redundancy
TIGR5
13,297
13,297
13,286
11
0
0.08%
13,021
12,042
979
276
7.51%
12,042
TCs
8,608
7,280
1,328
3,434
15.43%
12,042
TCs
10,836
8,657
2,179
1,206
20.10%
1,206
TCs
896
896
unknown
310
unknown
13,297
7,600
6,935 (TC)
665 (TC)
5,697
8.75% (TC)
(Enst)
(Enst)
Oligos
TIGR10
13,297
Oligos
Ensembl
Peptide
Ensembl
cDNA
Ensembl
HG DNA
GO
Annotation
Estimate
of
Redundancy
for SSWG124.17%
Oligos
(oligo
)
5,763
1,837
(Enst)
Microarray Analysis of Islet Quality and Function
•Diabetes Mellitus is world wide health problem
(1-2% of the population, 1 million U.S. citizens affected.)
•Type 1 diabetes mellitus - autoimmune destruction of the insulin
secreting pancreatic islet  cells, a disease of the young.
•Current management of diabetes involves daily blood sugar testing,
insulin injections, and careful meal planning.
•Insulin controls but doesn’t cure Diabetes.
•>40% of Diabetic Patients experience complications (can lead to
blindness, kidney failure, heart attacks and foot ulcers)
•The only way to CURE diabetes is to replace the destroyed beta cells
– Pancreas or Islet transplantation.
Islet Transplantation
Obligatory Ex-Vivo Culture
•Enzymatic Digestion to release islets
•Tissue Culture (48 Hours)
•Viability Assays
• Gene Expression Based Diagnostics?
Islets
Comprised
Islet
Cultureof
1000-2000
cells
Conditions
C
Cytokine
A: 5.6 mMol Glucose
 cells (70%) Insulin
B:(15%)
16.7 mMol
Glucose
 cells
Glucagon
 cells
C:(5%)
5.6 mMol Glucose
PP cells (5%)
+ (5%)
IL-1β (2ng/ml)
Endothelial
+ TNFα (1000U/ml)
+ IFNγ (1000U/ml)
D
A
B
D: 16.7 mMol Glucose
+ IL-1β (2ng/ml)
+ TNFα (1000U/ml)
+ IFNγ (1000U/ml)
Glucose
Intracellular ATP
ATP/DNA
(% of 5.6 mM glucose)
120%
100%
80%
60%
5.6 mM glucose
16.7 mM glucose
5.6 mM glucose + cytokines
16.7 mM glucose + cytokines
40%
20%
0
2
4
6
8
6
8
Time (Days)
Insulin content
Insulin/DNA
(% of 5.6 mM glucose)
120%
100%
80%
60%
5.6 mM glucose
16.7 mM glucose
5.6 mM glucose + cytokines
16.7 mM glucose + cytokines
40%
20%
0
2
4
Time (Days)
Pig Islet
Gene Expression
• Porcine Islet isolation and Culture:
• Islet cell donors: adult Landrace
sows
• Culture in medium 199 with
pig serum
• Culture condition: regular
medium 48 h, change to
conditional medium for 48 h
• RNA Isolation:
• Qiagen Rneasy mini kit
• RNA 6000 Nano LabChip Kit
Cell isolation
In vitro
culture
RNA isolation
Labeling
• Hybridization Protocol:
• Indirect labeling
• Data Acquisition:
• ScanArray 5000 (Scan)
Hybridization
Differentially expressed spots identified by microarray
analysis using GeneSpring and R/maanova methods
Increased
Experimental
Condition
Decreased
Control
Condition
GeneSpring
R/maanova
Overlap
GeneSpring
R/maanova
Overlap
1)
16.7 mM glucose
5.6 mM glucose
342
255
98
160
139
39
2)
16.7 mM glucose
+ cytokines
5.6 mM glucose +
cytokines
130
65
26
133
118
29
3)
5.6 mM glucose
+ cytokines
5.6 mM glucose
496
348
245
220
204
92
4)
16.7 mM glucose
+ cytokines
16.7 mM glucose
217
170
85
163
261
71
The number of spots with a p-value < 0.05 after pairwise examination are shown
A) Volcano Plots for Significant Genes
(LGLCvsLGNC)
B) Volcano Plots for Significant Genes
(HGLCvsHGNC)
GeneSpring
GeneSpring
6
6
4
GeneSpringOverlapped
R/Maanova
-Log(P-value)
-Log(P-value)
R/Maanova
4
GeneSpringOverlapped
2
2
R/MaanovaOverlapped
R/MaanovaOverlapped
0
-2
-1
0
0
1
2
3
-2
-1
Log2(Fold Change)
2
3
D) Volcano Plots for Significant Genes
(HGNCvsLGNC)
C) Volcano Plots for Significant Genes
(HGLCvsLGLC)
GeneSpring
GeneSpring
6
6
4
GeneSpringOverlapped
R/Maanova
-Log(P-value)
R/Maanova
-Log(P-value)
0
1
Log2(Fold Change)
4
GeneSpringOverlapped
2
2
R/MaanovaOverlapped
R/MaanovaOverlapped
0
0
-2
-1
0
1
Log2(Fold Change)
2
3
-2
-1
0
1
Log2(Fold Change)
2
3
Multifactorial Analysis of Expression Data




The mixed ANOVA models for pair-wise comparison, main effect and interaction
determination were:
ygijk =  + Di + Aj + Sk + gijk
(Pair-wise comparison)
ygijk =  + Di + Aj + Cg + Gg + gijk
(Main effect determination)
ygijk =  + Di + Aj + Cg + Gg + (CG)g + gijk
(Interaction determination)
 = mean value
D = dye effect
A = array
S = sample
C = cytokine
G = glucose
CG = interaction effect of glucose and cytokine
 = stochastic error
Gene Category
SSWG
1
Cytokine Upregulated
Cytokine Downregulated
Glucose Upregulated
Glucose Downregulated
Cytokine and Glucose
Interaction
biological_process
5791
210
179
89
87
91
response to external stimulus
590
62 (4.34E-15)
ns
ns
ns
16 (3.71E-02)
response to stress
413
30 (3.58E-04)
ns
ns
ns
ns
response to biotic stimulus
384
57 (8.94E-21)
ns
ns
ns
ns
immune response
317
52 (8.21E-21)
ns
ns
ns
ns
response to pest/pathogen/parasite
212
25 (4.72E-07)
ns
ns
ns
ns
82
11 (6.66E-04)
None
ns
ns
ns
humoral immune response
acute-phase response
11
4 (6.17E-03)
None
None
None
None
217
ns
13 (3.44E-02)
ns
ns
ns
complement activation
25
5 (1.15E-02)
None
None
ns
None
antigen processing
14
6 (8.95E-05)
None
None
None
ns
antigen presentation
13
5 (9.12E-04)
None
None
None
ns
cell death
221
17(5.90E-03)
ns
ns
ns
ns
apoptosis
207
16(7.53E-03)
ns
ns
ns
ns
vesicle-mediated transport
response to abiotic stimulus
196
ns
12 (3.83E-02)
ns
ns
ns
secretory pathway
85
ns
8 (1.51E-02)
ns
ns
ns
exocytosis
27
None
4 (4.86E-02)
None
ns
None
protein-membrane targeting
20
ns
4 (2.22E-02)
ns
ns
ns
nonselective vesicle transport
35
ns
ns
ns
None
4 (1.66E-02)
biosynthesis
699
ns
ns
19 (1.62E-02)
ns
ns
protein biosynthesis
373
ns
ns
12 (2.49E-02)
ns
ns
glucose metabolism
51
ns
ns
5 (7.22E-03)
ns
ns
catabolism
482
26 (4.00E-02)
ns
ns
ns
15 (1.59E-02)
macromolecule catabolism
352
23 (7.79E-03)
ns
ns
ns
ns
proteolysis and peptidolysis
328
22 (7.11E-03)
ns
ns
ns
ns
cell communication
1439
ns
ns
ns
ns
32 (2.55E-02)
signal transduction
1160
ns
ns
ns
ns
28 (1.52E-02)
cell adhesion
267
ns
16 (1.68E-02)
ns
ns
ns
cell migration
27
ns
5 (8.71E-03)
None
None
ns
morphogenesis
533
ns
28 (6.14E-03)
ns
ns
ns
organogenesis
475
ns
25 (1.00E-02)
ns
ns
ns
neuropeptide signaling pathway
41
ns
None
4 (2.25E-02)
ns
transcription from Pol II promoter
246
ns
17 (1.57E-02)
ns
ns
ns
None
Based on Eizirik et al, 2003
with blue ellipses were differentially
expressed both in response to
glucose alone and to cytokines alone.
Pig EST Cluster Statistics
ESTs downloaded from GenBank
Remaining ESTs after filtering for vector and E. coli
ESTs that matched proteins
398,794
396,897
214,776
Remaining ESTs after removing 729 putative
chimeras
Tentative unique transcripts (UT)
214,047
34,908
Contigs
19,798
Singletons
15,110
UT that matched proteins (non-chimeric)
33,238
Unique gene segments (UGS; splice variants grouped 27,967
by gene)
Protein families containing Pig ESTs
13,007
Acknowledgements
Fahrenkrug Lab
Hehuang Xie
Min Wang
Bhupinder Junej
Murtaugh Lab
Cheryl Dvorak
Hering Lab
Maria Hardstedt
Open Resources
TIGR
R/maanova
ENSEMBL