Optimization of the Affymetrix GeneChip Mapping 10K 2.0 Assay for routine clinical use on formalin-fixed paraffin-embedded tissues.
Author(s): Lyons-Weiler M, Hagenkord J, Sciulli C, Dhir R, Monzon FA
Publication: Diagn Mol Pathol, 2008, Vol. 17, Page 3-13
PubMed ID: 18303412 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to compare SNP call rates and comparative genomic hybridization (CGH) results between FFPE and OCT-embedded frozen renal tumors and between normal adjacent adrenal tissue and adrenal tumors. DNA was extracted from OCT embedded frozen tissue which had been scraped into RNAlater-ICE and from deparaffinized FFPE tissue scraped into lysis buffer using DNeasy with modifications.
Summary of Findings:
The authors report increasing the proteinase K digestion time from 1 day to 3 days did not increase the DNA yield. Increasing the amount of DNA used from 250 ng to 1 ug for the restriction digest step of template production led to a wider variety of fragment sizes, and the profile generated using 1 ug DNA from FFPE specimens was similar to that using 250 ng DNA from frozen specimens. The authors report increased template production when 1 ug of DNA from FFPE tissue was digested with 40 units of Xba I for 16 hours in 30 uL buffer. The template yield was not affected by adapter concentration, but yields and call rates were increased when 45 cycles were used instead of 40 or 35 cycles to amplify DNA from FFPE specimens. However, yields and call rates were lower and product sizes were smaller using the optimized protocol with DNA from FFPE specimens compared to using DNA from frozen specimens and a standard protocol. Call rates were higher using DNA from frozen rather than FFPE specimens and using normal adjacent rather than tumor tissue. 99.45% agreement in SNP calls was observed between frozen normal adjacent and tumor tissue and between replicates, and 95.98% agreement in SNP calls was observed between matched frozen and FFPE specimens. While LOH likelihood plots were comparable between FFPE and frozen specimens, small differences in copy number plots were observed. Copy number changes using the SNP array were confirmed by array CGH to bacterial artificial chromosome (BAC) arrays and microsatellite analysis.
Biospecimens
Preservative Types
- Formalin
- OCT
- RNAlater
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Normal Adjacent
- Neoplastic
Platform:
Analyte Technology Platform DNA Array CGH DNA DNA microarray DNA Microsatellite analysis DNA Automated electrophoresis/Bioanalyzer DNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
OCT
DNA microarray Specific Nucleic acid amplification 35 cycles
40 cycles
45 cycles
DNA microarray Specific Template/input amount 250 ng
500 ng
1 ug
Analyte Extraction and Purification Protein digestion 1 day
3 days
DNA microarray Specific Template modification 2 x adapter
4 x adapter
2.5 h Xba digest
16 h Xba digest
Array CGH Specific Technology platform Affymetrix 10k Xba arrays
BAC arrays
Microsatellite analysis