Download Normal Variation MIAME Information

11-01-06 MIAME Checklist for Normal Variation Paper
Experiment Design:
 The goal of the experiment – one line maximum (e.g., the title from the
related publication)
Title: Genetic background influences murine prostate gene expression:
Associations with cancer phenotypes in man.
 A brief description of the experiment (e.g., the abstract from the related
publication):
Cancer of the prostate is influenced both by genetic predisposition and
environmental factors. The identification of genes capable of modulating
cancer development has the potential to unravel disease heterogeneity and aid
diagnostic and prevention strategies through improved understanding of geneenvironment interactions. To this end, mouse models have been developed to
isolate the influences of individual genetic lesions in the context of consistent
genotypes and environmental exposures. However, the extent of normal
prostatic phenotypic variability dictated by a genetic background potentially
capable of influencing the process of carcinogenesis has not been established.
In this study we used microarray analysis to quantitate transcript abundance
levels in the prostates of five commonly studied inbred mouse strains. We
applied a multiclass response t-test to identify genes whose expression in each
strain significantly differed from the other four strains. Approximately 13%
(932 genes) exhibited significant differential expression (range 1.3 to 190fold) in one strain relative to other strains (FDR≤10%). The pattern of
variability in transcript levels did not result from variations unique to a
particular strain, but rather represented genetic variability across all five
strains assessed. Expression differences were confirmed by qRT-PCR, or
immunohistochemistry for several genes previously shown to influence cancer
progression such as Psca, Mmp7, and Clusterin. Analyses of human prostate
transcripts orthologous to variable murine prostate genes identified differences
in gene expression in benign epithelium that correlated with the differentiation
state of adjacent tumors. For example, the expression of apolipoprotein D, a
gene known to enhance resistance to cell stress in Drosophila, was expressed
at significantly greater levels in benign epithelium associated with high-grade
versus low-grade cancers. These data support the concept that the cellular,
tissue, and organismal context contribute to oncogenesis and suggest that a
predisposition to a sequence of events leading to pathology may be
determined prior to cancer initiation.
 Keywords, for example, deletion, gain, amplification, polymorphisms
detection, strain determination (the use of MGED ontology terms is
recommended):
Keywords: mouse, prostate, transcript, variation, microarray, cancer,
apolipoprotein D

Experimental factors - the parameters or conditions tested, for example,
disease state, strain, species isolate (the use of MGED ontology terms is
recommended):
Genetic variation

Experimental design - relationships between samples, treatments, extracts,
labeling, and arrays (e.g., a diagram or table)
Experimental design. Prostates from twelve mice from each of five strains of
Mus musculus: C57BL/6J, 129X1/SvJ, BALB/cAnNCrl, FVB/NJ and
DBA/2NCrl, were resected and individual lobes were dissected: DP: dorsal
prostate; LP: lateral prostate; VP: ventral prostate; AP: anterior prostate. Each
experimental sample represents a pool of equal amounts of RNA for each
prostatic lobe from 3 animals. Four independent experimental samples were
created per strain: 12 mice divided into 4 pools of 3 mice each for a total of 4
microarray experiments per strain. Amplified RNA from each experimental
sample was hybridized against a reference sample (created by combining
equal amounts of RNA from all the samples from all strains) onto custom
mouse prostate cDNA microarrays using alternate dye-labeling to account for
dye-specific effects.

Quality control steps taken (e.g., replicates or dye swaps):
1) 12 mice, 4 pools of 3 mice per strain for a total of 4 microarray
experiments per strain
2) Half of the biological experimental samples per strain were labeled with
Cy3 dye and the reference with Cy5 dye, and in the other half the labeling was
inversed.

Links to the publication, any supplemental websites or database accession
numbers:
http://www.mpedb.org/SUPPLEMENTARY/SUBMITTED/DBIANCHI
Samples used, extract preparation and labelling:
 The origin of each biological sample (e.g., name of the organism, the provider
of the sample) and its characteristics (e.g., gender, age, developmental stage,
strain, or disease state). This information must not compromise the patients
anonymity – legal and ethical requirements take the precedence.
Table 1. Mouse strain
Strain
129X1/SvJ
C57BL/6J
FVB/NJ
DBA/2NCrl
BALB/cAnNCrl
Vendor
Jackson Labs
Jackson Labs
Jackson Labs
Charles River
Charles River
Stock number
691
664
1800
026
028
Sex
MALE
MALE
MALE
MALE
MALE
Age
7 weeks
7 weeks
7 weeks
7 weeks
7 weeks
Table 2. Experimental samples
#
Sample
pool ID
Strain
Lobe
Age
Pool Components
1
129-1
129X1/SvJ
DP, LP, VP and AP
8-9 weeks
129X1/SvJ; mouse # 1, 5 and 12
2
129-2
129X1/SvJ
DP, LP, VP and AP
8-9 weeks
129X1/SvJ; mouse # 2, 7 and 11
3
129-3
129X1/SvJ
DP, LP, VP and AP
8-9 weeks
129X1/SvJ; mouse # 3, 6 and 9
4
129-4
129X1/SvJ
DP, LP, VP and AP
8-9 weeks
129X1/SvJ; mouse # 4, 8 and 10
5
Balb-1
BALB/cAnNCrl
DP, LP, VP and AP
8-9 weeks
BALB/cAnNCrl; mouse # 1, 5 and 12
6
Balb-2
BALB/cAnNCrl
DP, LP, VP and AP
8-9 weeks
BALB/cAnNCrl; mouse # 2, 7 and 10
7
Balb-3
BALB/cAnNCrl
DP, LP, VP and AP
8-9 weeks
BALB/cAnNCrl; mouse # 3, 6 and 11
8
Balb-4
BALB/cAnNCrl
DP, LP, VP and AP
8-9 weeks
BALB/cAnNCrl; mouse # 4, 8 and 9
9
C57-1
C57BL/6J
DP, LP, VP and AP
8-9 weeks
C57BL/6J; mouse # 1, 6 and 11
10
C57-2
C57BL/6J
DP, LP, VP and AP
8-9 weeks
C57BL/6J; mouse # 2, 7 and 10
11
C57-3
C57BL/6J
DP, LP, VP and AP
8-9 weeks
C57BL/6J; mouse # 3, 4 and 9
12
C57-4
C57BL/6J
DP, LP, VP and AP
8-9 weeks
C57BL/6J; mouse # 5, 8 and 12
13
DBA-1
DBA/2NCrl
DP, LP, VP and AP
8-9 weeks
DBA/2NCrl; mouse # 1, 8 and 11
14
DBA-2
DBA/2NCrl
DP, LP, VP and AP
8-9 weeks
DBA/2NCrl; mouse # 2, 5 and 12
15
DBA-3
DBA/2NCrl
DP, LP, VP and AP
8-9 weeks
DBA/2NCrl; mouse # 3, 6 and 9
16
DBA-4
DBA/2NCrl
DP, LP, VP and AP
8-9 weeks
DBA/2NCrl; mouse # 4, 7 and 10
17
FVB-1
FVB/NJ
DP, LP, VP and AP
8-9 weeks
FVB/NJ; mouse # 1, 5 and 12
18
FVB-2
FVB/NJ
DP, LP, VP and AP
8-9 weeks
FVB/NJ; mouse # 2, 7 and 11
19
FVB-3
FVB/NJ
DP, LP, VP and AP
8-9 weeks
FVB/NJ; mouse # 3, 6 and 9
20
FVB-4
FVB/NJ
DP, LP, VP and AP
8-9 weeks
FVB/NJ; mouse # 4, 8 and 10



Manipulation of biological samples and protocols used (e.g., stress, drugs).
NA
Experimental factor value for each experimental factor, for each sample (e.g.,
‘disease state = AML’ for a sample in a time course experiment).
Strain differences
Technical protocols for preparing the hybridization extract and labeling:
Prostate tissues were homogenized using a Polytron and total RNA was
isolated using the RNeasy extraction kit (Qiagen). Equal amounts of total RNA
(0.7 µg) from each of the four pooled lobes/strain were combined to produce
secondary pools, and amplified with the MessageAmpTM aRNA kit (Ambion).
Amplified RNA from each secondary pool (four pools per strain) was hybridized
separately to cDNA microarrays for a total of 4 independent biological replicates
per strain (12 mice, 4 pools of 3 mice per strain for a total of 4 microarray
experiments per strain; see Figure 1). A common reference sample was created by
combining equal amounts of RNA from all the samples from all strains.
The amplified RNA was used as template for cDNA probe synthesis
followed by hybridization to a custom mouse prostate cDNA array (mPEDB
array), comprised of ~8300 genes expressed in the developing and adult mouse
prostate (http://www.mpedb.org/). The 20 microarrays used for the experiment
were all from the same printing batch. The protocol used for indirect labeling of
cDNAs was a modification of a method described elsewhere
(http://cmgm.stanford.edu/pbrown/protocols/aadUTPCouplingProcedure.htm).
Briefly, cDNA probes were made from 2 µg of amplified RNA in a reaction
volume of 30 µl containing 170 ng random hexamer primers, 0.2 mM 5-(3aminoallyl)-2_-deoxyuridine-5_-triphosphate (amino acid-dUTP; Sigma-Aldrich),
0.3 mM dTTP, 0.5 mM each dATP, dCTP, and dGTP, and 380 units of
Superscript II reverse transcriptase (Life Technologies) incubated at 42oC for 120
minutes. After RNA hydrolysis, purified cDNA was combined with either Cy3 or
Cy5 monoreactive fluors (Amersham Pharmacia) that covalently couple to the
cDNA incorporated aminoallyl linker in the presence of 50 mM NaHCO3 (pH
9.0). The coupling reaction was quenched with Hydroxylamine and reference and
experimental probes were combined, filtered, and competitively hybridized to
microarrays under a coverslip for 16 h at 63°C. Slides were washed sequentially
with 1X saline sodium citrate (SSC)/0.03% sodium dodecyl sulfate (SDS), 1X
SSC, 0.2X SSC, 0.05X SSC, and spun dry. To account for dye bias, half of the
biological experimental samples per strain were labeled with Cy3 dye and the
reference with Cy5 dye, and in the other half the labeling was inversed.
Hybridization procedures and parameters:

The protocol and conditions used for hybridization, blocking and washing,
including any post-processing steps such as staining.
Reference and experimental probes were combined, filtered, and competitively
hybridized to microarrays under a coverslip for 16 h at 63°C. Slides were washed
sequentially with 1X saline sodium citrate (SSC)/0.03% sodium dodecyl sulfate
(SDS), 1X SSC, 0.2X SSC, 0.05X SSC, and spun dry.
Measurement data and specifications:

Data
o The raw data, i.e. scanner or imager and feature extraction output
(providing the images is optional). The data should be related to the
respective array designs (typically each row of the imager output should
be related to a feature on the array – see Array Designs).
Table 3. gpr file information
Sample
pool ID
gpr file name
GSM Number
GenePix Results #
GenePix
Version
Gal File Used
129-1
MuPr0294_129-1_ref_20040722.gpr
GSM138319.gpr
GenePix Results 3
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
129-2
MuPr0286_ref_129-2_20040722.gpr
GSM138315.gpr
GenePix Results 3
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
129-3
MuPr0293_ref_129-3_20040722.gpr
GSM138318.gpr
GenePix Results 3
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
129-4
MuPr0290_129-4_ref_20040722.gpr
GSM138317.gpr
GenePix Results 3
Balb-1
MuPr0225_ref_Balb-1_20040607.gpr
GSM138300.gpr
GenePix Results 3
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
Balb-2
MuPr0240_Balb-2_ref_20040611.gpr
GSM138306.gpr
GenePix Results 3
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
Balb-3
MuPr0251_Balb-3_ref_20040611.gpr
GSM138313.gpr
GenePix Results 3
Balb-4
MuPr0250_ref_Balb-4_20040611.gpr
GSM138312.gpr
GenePix Results 3
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
C57-1
MuPr0228_ref_C57-1_20040607.gpr
GSM138302.gpr
GenePix Results 3
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
C57-2
MuPr0288_C57-2_ref_20040722.gpr
GSM138316.gpr
GenePix Results 3
C57-3
MuPr0241_C57-3_ref_20040611.gpr
GSM138307.gpr
GenePix Results 3
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
C57-4
MuPr0244_ref_C57-4_20040611.gpr
GSM138309.gpr
GenePix Results 3
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
DBA-1
MuPr0229_ref_DBA-1_20040607.gpr
GSM138303.gpr
GenePix Results 3
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
DBA-2
MuPr0236_DBA-2_ref_20040611.gpr
GSM138305.gpr
GenePix Results 3
DBA-3
MuPr0234_DBA-3_ref_20040611.gpr
GSM138304.gpr
GenePix Results 3
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
DBA-4
MuPr0249_ref_DBA-4_20040611.gpr
GSM138311.gpr
GenePix Results 3
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
FVB-1
MuPr0226_ref_FVB-1_20040607.gpr
GSM138301.gpr
GenePix Results 3
FVB-2
MuPr0254_FVB-2_ref_20040611.gpr
GSM138314.gpr
GenePix Results 3
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
FVB-3
MuPr0243_FVB-3_ref_20040611.gpr
GSM138308.gpr
GenePix Results 3
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
GSM138310.gpr
GenePix Results 3
GenePix Pro 4.1
05-10-04 MPEDB_401-800.gal
FVB-4
MuPr0246_ref_FVB-4_20040611.gpr
o The normalized and summarized data, i.e., set of quantifications from
several arrays upon which the authors base their conclusions (for gene
expression experiments also known as gene expression data matrix and
may consist of averaged normalized log ratios). The data should be related
to the respective array designs (typically each row of the summarized data
will be related to one biological annotation, such as clone name or specific
oligo).
See Normalized Log Ratios File.

Data extraction and processing protocols,
o Image scanning hardware and software, and processing procedures and
parameters. : Fluorescent array images were collected for both Cy3 and
Cy5 using a GenePix 4000B fluorescent scanner (Axon Instruments,
Foster City, CA). The image intensity data were gridded and extracted
using GenePix Pro 4.1 software.
o Normalization, transformation and data selection procedures and
parameters. For each array spot, the expression levels of the two
fluorophores were obtained by subtracting median background intensity
from median foreground intensity. A gene was only considered expressed
if the fluorescence intensity of the corresponding spot was at least 6
foreground pixels greater than 4 standard deviations above background in
at least half of the arrays per strain. Array spot not meeting these criteria
were designated NA. For each gene, the logarithm base 2 ratios (referred
henceforth as log ratios) of the two channels were calculated to quantify
the relative expression levels of genes between the experimental and
reference samples. To allow for inter-array comparisons, each array was
normalized to remove systematic sources of variation. This was
accomplished by means of a print-tip-specific intensity-based
normalization method [35]. A scatter-plot smoother, which uses robust
locally linear fits, was applied to capture the dependence of the log ratios
on overall log-spot intensities. The log ratios were normalized by
subtracting the fitted values based on the print-tip-specific scatter-plot
smoother from the log ratios of experimental and control channels. After
normalization, spots were removed from further analysis if they had more
then 3 NA values or if they were in the lower third quantile of abundance
across all 20 arrays.