Ovarian carcinomas with genetic and epigenetic BRCA1 loss havedistinct molecular abnormalities Page: 4 of 12

                                
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gene of interest are gained relative to MYOD 1 in any given
sample. PCR experiments were carried out in a volume of
10  L with 384-well plates and an Applied Biosystems
7900 HT Sequence Detector (Applied Biosystems, Foster
City, CA). The fluorescence signal of the quantitative
methylation-specific PCR was generated by SYBR Green I.
Samples (10 ng bisulfite-treated DNA) were run in tripli-
cate containing 5  L SYBR Green Master Mix (Applied
Biosystems, Foster City, CA) and 5 pmol of each forward
and reverse primer. Bisulfite-converted CpG methylated
Jurkat Genomic DNA (New England Biolabs, Ipswich,
MA) served as a positive control and was used to generate
a standard curve to quantify the amount of fully methyl-
ated promoters in each reaction. PCR amplification was
done by means of the following procedure: 95 C for 15
minutes, followed by 40 cycles at 95'C for 15 seconds,
62 C for 1 minute. A subsequent dissociation curve anal-
ysis checked the specificity of products. FANCF promoter
hypermethylation was assessed using a HpaII digest assay
and  methylation-specific PCR  protocol previously
reported by Taniguchi et al [52].
Real-time Q-RT-PCR
Extracted RNA was treated with DNAse I (Invitrogen,
Carlsbad, CA) prior to creating cDNA using random hex-
amer priming and MMLV reverse transcriptase (Invitro-
gen, Carlsbad, CA). Applied Biosystems Taqman primer/
probe kits (Hs00173233_ml (BRCA1), Hs00609060_ml
(BRCA2),          Hs01920652_si           (PTEN),
Hs00907966_m1(PIK3CA)) were used to quantify mRNA
expression levels using real-time qRT-PCR [37] and an ABI
Prism 7900 HT Sequence Detection System (Applied Bio-
systems, Foster City, CA). Relative gene expression quan-
tification was calculated according to the comparative Ct
method using human 18S ribosomal RNA (Applied Bio-
systems, Foster City, CA) and commercial RNA controls
(Stratagene, La Jolla, CA). Relative quantification was
determined as follows: 2-(Act sample-Act calibrator). Ratios
(tumor relative gene expression:average of all tumors) less
than 0.7 or greater that 1.3 for were scored as decreased or
increased mRNA expression, respectively.
Immunohistochemistry
The BRCA1 antibody Ab-1 (Oncogene, 1:50 dilution) was
used and antigen retrieval was performed in ix EDTA
buffer (pH 8.0) by microwaving for 2 minutes, and then
boiling in a waterbath for 30 minutes. Endogenous perox-
ide activity was blocked with 3% hydrogen peroxide and
then sections were incubated with 2.5% normal horse
blocking serum. Following incubation with the primary
antibody, the Vector Laboratories (Burlingame, CA)
ImmPRESS kit was used according to the manufacturer's
recommendations to visualize antibody complexes.
Nuclear staining was assessed by CBG, who was blinded
to all other BRCA analysis. Tumors were considered

BRCA1 positive if greater than 1% of tumor nuclei showed
staining. IHC was also performed with the following
panel of previously validated antibodies using a Ventana
(Tucson, AZ) automated immunostainer: p21 (Neomark-
ers, Fremont, CA, clone DCS-60.2, 1:100 dilution), p53
(Dako, Carpinteria, CA, clone DO-7, 1:400 dilution), and
WT-1 (Dako, Carpinteria, CA, clone 6F-H2, 1:50 dilu-
tion). BRCA1 IHC was done on whole sections, while
other IHC markers were assessed using sections from a tis-
sue microarray constructed with two 0.6 mm cores per
case.
Molecular Inversion Probe (MIP) Copy Number
The MIP copy number assay was done as described previ-
ously [53] with some modifications. Specifically, the cur-
rent protocol is a modification   of the Targeted
Genotyping protocol commercialized by Affymetrix [54].
Test DNA samples were diluted toi6 ng/ l. Molecular
inversion probes were annealed to DNA by mixing 4.7  d
of DNA (75 ng total), 0.75  l of Buffer A, 1.1  l of the 53
K molecular inversion probe pool (200 amol/ l/probe)
and 0.045  l of Enzyme A in a 384-well plate on ice. The
reaction was incubated for 4 min at 20 C, 5 min at 95 C,
then overnight at 58'C. Following annealing, 13  l of
Buffer A and 1.25  l of Gap Fill Enzyme mix were added
to each reaction and 9  l of reaction volume was trans-
ferred to each of two wells in a 96-well plate. Molecular
inversion probes were circularized with 4  l of dNTP mix
at 58 C for 10 min. Linear probes and genomic DNA were
eliminated by addition of 4  l of Exo Mix and incubation
at 37 C for 15 min, followed by universal primer ampli-
fication for 18 cycles (20 sec at 95'C, 40 sec at 64'C, and
10 sec at 72 C). For labelling reactions, the product was
further amplified for 10 cycles using labelled primers,
then subjected to cleavage by HY Digest Mix at 37'C for 2
hours. The cleaved MIP products were mixed with Hybrid-
ization Cocktail, denatured, and hybridized to 70 K Uni-
versal Taq arrays at 39 C for 16 h (two arrays per sample).
The overnight hybridized arrays were washed on a Gene-
Chip Fluidics Station FS450 and stained by SAPE at 5 ng/
ml (Invitrogen).
Copy number estimation was obtained from the hybridi-
zation signals as previously described [55], with the fol-
lowing modifications: the color-separation step was
omitted as the single color readout on two arrays pre-
vented the spectral overlap that occurs with multi-color
readouts, and Langmuir correction was performed instead
of linear calibration of allele signals [56]. Copy numbers
over 3.0 were considered amplification events and copy
numbers below 1.5 were considered deletion events.
Data analysis
Epigenetic BRCA1 loss was defined as having promoter
hypermethylation accompanied by either low relative
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BMC Cancer 2008, 8:17