Optimization of the Affymetrix GeneChip Mapping 10K 2.0 Assay for routine clinical use on formalin-fixed paraffin-embedded tissues.
The use of chromosomal copy number changes as markers for tumor behavior or as prognostic markers for patient outcome has been suggested. However, current clinically used technologies cannot perform genome-wide assessment of chromosome copy number and analysis of loss of heterozygosity in the same assay for paraffin-embedded tissue. We have optimized the Affymetrix GeneChip Mapping Assay for the 10K 2.0 array for use with formalin-fixed, paraffin-embedded (FFPE) tissues. This technology uses single nucleotide polymorphism (SNP) arrays to assess the changes in chromosomal copy number and loss of heterozygosity. DNA from 3 paired tumor/normal samples of adrenal tumors and 4 samples of renal tumors were processed with modifications to the manufacturer's protocol. Modifications at different steps were evaluated for their effects on SNP signal-detection and call rates. Frozen samples showed 99.6%+/-0.3% signal-detection rates and 94.7%+/-3.0% SNP call rates. FFPE samples labeled with the original protocol failed to produce enough polymerase chain reaction products for hybridization, whereas the same samples processed with the optimized protocol had signal-detection rates of 97.4%+/-0.018% and SNP call rates of 90.9%+/-0.034%. The average SNP call concordance between fresh and matching FFPE samples was 96%. Chromosomal aberrations detected in the frozen tumors were also detected in the FFPE tissues. Our optimized protocol significantly improves the performance of the FFPE samples in the Affymetrix GeneMapping Assay with the 10K 2.0 SNP array. This optimized protocol opens up the potential for the GeneChip Mapping assay to be used in the development of clinical test assays.
Lyons-Weiler
Maureen
M
Clinical Genomics Facility, University of Pittsburgh, Pittsburgh, PA, USA.
Hagenkord
Jill
J
Sciulli
Christin
C
Dhir
Rajiv
R
Monzon
Federico A
FA
D000310:Adrenal Gland Neoplasms / Q000175:diagnosis / Q000235:genetics; D002138:Calibration; D002874:Chromosome Mapping / Q000379:methods; D002877:Chromosomes, Human; D005557:Formaldehyde / Q000494:pharmacology*; D005629:Frozen Sections; D018628:Gene Dosage; D006801:Humans; D007680:Kidney Neoplasms / Q000175:diagnosis / Q000235:genetics; D019656:Loss of Heterozygosity; D016555:Matched-Pair Analysis; D009375:Neoplasms, Glandular and Epithelial / Q000175:diagnosis / Q000235:genetics; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000592:standards*; D016612:Paraffin Embedding; D010338:Pathology, Clinical / Q000379:methods*; D010673:Pheochromocytoma / Q000175:diagnosis / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D046888:Tissue Array Analysis / Q000379:methods*; D016707:Tissue Fixation
D005557:Formaldehyde