MIAME COMPLIANCE CHECKLIST:
Kennerly, Thompson, Olby, Breen, Gibson (2004)
Comparison of Regional Gene Expression Differences in the Brains of the Domestic Dog
and Human
Experiment Design
Type of Experiment:
Quantitative analysis of regional gene expression
differences in brains of canines
Experimental Factors:
Three dog breeds: 3 American Staffordshire Terriers, 1
Beagle, and 1 American Foxhound
Three brain regions: Cortex, Cerebellum, and Pituitary
Gland
Number of Hybridizations:
30
Type of Reference:
None
Hybridization Design:
One complete main loop, where each like tissue was
compared across different breeds, and five smaller
loops, where tissue types from a single dog were
compared to the other tissue types in the same dog.
Quality Control:
Each of the 15 samples (5 dogs with 3 tissue types) with
multiple RNA amplifications were pooled.
Each RNA sample was split into 4 separate cDNA
synthesis reactions (15x4 = 60), these were then
pooled and re-split into four separate tubes (two for
each labeling reaction).
Each comparison of samples were balanced for the two dye
types
Supplementary Data URL:
http://statgen.ncsu.edu/ggibson/SupplInfo/SupplInfo9.htm
Samples used, Extract preparation and labeling:
Biological Samples:
Canis Familiaris (Canine)
5 purebred canine dogs: 3 American Staffordshire Terriers,
1 Beagle, and 1 American Foxhound
Cerebellum, cortex, and pituitary gland tissue from each
canine were excised under sterile conditions during
necropsy
All dogs submitted to NCSU Veterinary Teaching Hospital
for euthanasia and necropsy at owners request
Biological Manipulations:
All tissues were flash frozen immediately upon removal in
liquid nitrogen and stored at –80C
RNA was extracted using TRIzol manufactures protocol
(Invitrogen), run through Qiagen RNeasy Mini Kit
for clean up, and stored at –80C
RNA was linear amplified using Agilent Low Input
Fluorescent Linear Amplification Kit (5184-3523)
*Protocol was modified by only performing a
single round of amplification to get amplified
complementary RNA
Labeling Protocols:
Labeled amplified RNA samples with Cy3 or Cy5 using
aminoallyl indirect labeling protocol from The
Institute for Genomic Research (SOP#M004)
External Controls:
None
Hybridization procedures and parameters:
Hybridization protocol:
Hybridizations were performed for 20 hours at 42C. All
hybridizations were performed between August 6,
2003 and September 20, 2003.
Washing Protocol:
Washings were performed using a standard series of three
high stringency washes
Measurement data and specifications:
Scanning and software:
ScanArray 4000 Microarray Analysis System Scanner
(Packard Bioscience).
Sixty (two for each array) acquired TIFF images were
analyzed using ScanAlyze 2
(http://rana.lbl.gov/Eisensoftware.htm)
Data Files:
30 raw data files in text format
1 data file for SAS import with log2 values for each array,
with spots at background or below removed
Type of Data:
Raw fluorescence intensity values with background values
subtracted
Log base 2 transformations of raw subtracted fluorescence
values
Data Transformation:
Subtracted background average from the spot raw intensity
levels for each dye channel
Averaged log2 values for each spot across all arrays and
removed spots with average value at or below
background levels
2 -Step Mixed Model ANOVA:
-Step 1: Compute relative fluorescence intensities
(RFI), by removing overall dye and array
effects
-Step 2: Evaluate breed specific effects, tissue specific
effects and breed and tissue interaction
effects.
Volcano plots were used to evaluate the significance of
expression against the magnitude of the expression
(plotted as negative log p-value against log base 2
value)
Two way hierarchical clustering was done to evaluate
regional expression in the brain
Final Gene Expression Data:
Worksheet 1: Selected Gene list with annotations
Worksheet 2: Results of Mixed Model ANOVA
Worksheet 3: Raw Data file
Link:
http://statgen.ncsu.edu/ggibson/SupplInfo/SupplInfo9.htm
Array Design:
General:
cDNA arrays printed with a pin and loop printer on amino
propyl silane coated slides (Corning Gaps II)
UV Crosslinking binds DNA to the slide