MIAME Checklist for Affy 10K SNP bladder:
Experiment design:
 Type of experiment: Comparison between germline (blood) and microdissected bladder
tumors to determine allelic imbalances.
 Experimental factors: Determine the loss of heterozygous SNPs in tumor DNA and correlate
the lost SNPs to the chromosomal locations.
 The number of hybridizations performed in the experiment: 37 microdissected bladder
tumors from 17 patients and 17 blood samples from the same patients. 54 hybridizations.
 Hybridization design: the number of heterozygous SNPs in DNA from blood was compared
to the number of heterozygous SNPs in DNA from tumor in the same patient.
 Quality control steps taken: The arrays were checked for incompatible SNP calls/Mendelian
Inheritance Errors (e.g. homozygous AA in blood converted to homozygous BB in tumor
from the same patient). None found.
Samples used, extract preparation and labeling:
 The origin of the biological samples and its characteristics: blood and bladder tumor tissue
from Homo Sapiens.
Patient ID- visit number
112-2
112-12
747-3
747-5
825-3
825-5
865-1
865-2
1058-3
1058-10
140-8
140-12
140-16
154-5
154-6
154-10
166-5
166-9
166-14
941-4
941-6
172-3
172-4
365-1
365-3
501-1
501-5
839-1
839-2
1013-1
1013-3
1017-1
1017-3
1033-1
1033-2
1276-1
1276-3
Time between tumors (months)
47
7
6
5
29
18
15
10
31
25
24
10
5
7
26
12
9
9
6
7
Stage
Ta
T1
Ta
T1
Ta
T1
Ta
T1
Ta
T1
Tumor
Grade
Gr. III
Gr. III
Gr. II
Gr. III
Gr. III
Gr. III
Gr. II
Gr. II
Gr. II
Gr. III
Ta
Ta
T1
Ta
Ta
T1
Ta
Ta
T1
Gr. III
Gr. III
Gr. III
Gr. II
Gr. II
Gr. III
Gr. II
Gr. II
Gr. III
Ta
T2-4
T1
T2-4
T1
T1/T2-4
T1
T2-4
T1
T2-4
T1
T2-4
T1
T2-4
T1
T2-4
T1
T1/T2
Gr. III
Gr. III
Gr. III
Gr. III
Gr. III
Gr. III
Gr. III
Gr. III
Gr. III
Gr. III
Gr. III
Gr. III
Gr. III
Gr. III
Gr. III
Gr. III
Gr. III
Gr. III


Manipulation of biological samples and protocols used: Tumors for DNA extraction were
frozen immediately after surgery and stored at –80°C. Prior to DNA extraction the tumors
were transferred to Tissue-Tek® (Sakura Finetek, Tokyo, Japan) and tumor tissue was
crudely microdissected from tumor sections (20 µm). Germline DNA was purified from
blood from the same patient.Protocol for preparing the hybridization extract: DNA was
extracted from tumor tissue and blood using a PUREGENE® protocol (Gentra SYSTEMS,
Minneapolis, MN) according to the manufacturer’s instructions
(http://www.gentra.com/products.asp?product_family_ID=1 ). Basically the purification
protocol for tumor tissue is: add tumor tissue to a 1.5 ml centrifuge tube containing 300 µl
Cell Lysis Solution and 5-6 µl Proteinase K (5 mg/ml). Mix and incubate at 55°C for 3
hours to overnight, until tissue particulates have dissolved. Cool sample to room
temperature and add 100 µl Protein Precipitation Solution to the cell lysate. Vortex
vigorously. Centrifuge at 13,000-16,000 x g for 3 minutes. Pour the supernatant containing
the DNA into a clean 1.5 ml centrifuge tube containing 300 µl 100% Isopropanol (2propanol). Mix the sample by inverting. Centrifuge at 13,000-16,000 x g for 1 minute. Pour
off supernatant and drain tube on clean absorbent paper. Add 300 µl 70% Ethanol and wash
the DNA pellet. Centrifuge at 13,000-16,000 x g for 1 minute. Pour off the ethanol. Air dry
10-15 minutes. Add 25-100 µl DNA Hydration Solution. Rehydrate DNA by incubating
sample 1 hour at 65ºC and/or overnight at roomtemperature. The DNA concentration was
determined using a spectrophotometer, and diluted to a concentration of 50 ng/µl.
Labeling protocol: The Single Primer Assay Protocol (preparation of DNA target, labeling,
hybridization to 10K GeneChip® Early Access, washing and staining) was performed
according to the manufacturer’s instructions (Affymetrix, Santa Clara, CA
https://www.affymetrix.com/support/downloads/manuals/10k_manual.pdf ). In short, 250 ng
of genomic or tumor DNA was digested with 10 Units of Xba1. The 4 bp Xba1 overhangs
were ligated (T4DNA Ligase) to an Adaptor Xba fragment. A PCR reaction was set up with
a PCR primer complementary to the Adaptor Xba fragment (PCR primer Xba). For each
sample 4-5 PCR reactions were needed. The PCR products were purified according to the
Qiagen manual (QIAquick PCR Purification Kit Protocol, Qiagen, Darmstadt, Germany)
except that all DNA elutes from the 4-5 PCR reactions were collected in one tube. 20 µg of
purified PCR product was fragmentized with DNAse. The fragmented DNA was then endlabeled by biotinylated-ddATP in the presence of ~30 U Terminal Transferase (TdT).
Hybridization procedures and parameters:
 The labeled DNA was hybridized to the 10K chip overnight. After removal of the
hybridization cocktail, the chip was stained with streptavidin for 45 minutes, then with
biotinylated Anti-Streptavidin for 10 minutes, and then with Streptavidin-R-phycoerythrin
conjugate, for 20 minutes.
Measurement data and specifications:
 The chips were scanned using the HP (Hewlett-Packard, Palo Alto, CA, USA) scanner
according to the manufacturer's recommended conditions (HuSNP Mapping Assay Manual,
Affymetrix PN 700308). Hybridization signal was detected by Affymetrix Microarray Suite
5.0 software (Affymetrix,). Genotype calls were generated using the Genotyping Tools
software.
 The SNPs were positioned according to the same genome build. The genome build used
was: UCSC Genome Browser http://genome.ucsc.edu/ april 2003 assembly. The April 2003
human reference sequence (UCSC version hg15) is based on NCBI Build 33. The data can
be found in the attached file: Affy 10K snp bladder.xls
Array Design:
 The used SNP detecting chip was: GeneChip® Mapping 10K Early Access Array
Affymetrix, Santa Clara, CA. Technical information can be found at the Affymetrix website:
http://www.affymetrix.com/support/technical/byproduct.affx?product=10k Basically, the
early access array has the same features as the GeneChip® Human Mapping 10K Array, but
as it was an early version a few of probes have been removed later due to poor performance.
 The SNPID refers to The SNP Consortium ID (TSC). Website: http://snp.cshl.org/