DNA amplification method tolerant to sample degradation.
Author(s): Wang G, Maher E, Brennan C, Chin L, Leo C, Kaur M, Zhu P, Rook M, Wolfe JL, Makrigiorgos GM
Publication: Genome Res, 2004, Vol. 14, Page 2357-66
PubMed ID: 15520297 PubMed Review Paper? No
Suggested by: ISBER
Purpose of Paper
The purpose of this paper was to determine the effects of amplifying genomic DNA from formalin-fixed paraffin-embedded (FFPE) gliomas using Restriction and Circularization-Aided Rolling Circle Amplification (RCA-RCA) on DNA analysis using real-time qPCR and comparative genomic hybridization (CGH).
Conclusion of Paper
Whole genome amplification (WGA) was successful using both multiple displacement amplification (MDA) and RCA-RCA techniques, but MDA also produced non-specific amplification artifacts when no DNA was present. RCA-RCA amplified DNA was generally of a high molecular weight (mostly above 10 kb), but with increasing storage of the FFPE specimen, the amplified fragment size reduced (0.5-4 kb in the specimen stored for 29 months). Real-time qPCR analysis revealed that RCA-RCA increased the amount of DNA by 600-4000 fold, but amplification amount depended on gene and storage duration. Generally, copy number, as determined by real-time qPCR, was similar in amplified and unamplifed DNA from FFPE gliomas, but when the DNA was highly degraded (stored 29 months), this correlation was lost. CGH profiles were similar in unamplified and RCA-RCA amplified DNA from FFPE specimens. In contrast, MDA amplified DNA was unsuitable for CGH.
Studies
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Study Purpose
The purpose of this study was to determine the effects of amplifying genomic DNA from FFPE gliomas using RCA-RCA on DNA analysis using real-time qPCR and CGH. Specimens were fixed for 7 h, and DNA was extracted using the Qiagen kit after 17-29 months of FFPE block storage.
Summary of Findings:
WGA was successful using both the MDA and RCA-RCA techniques, but MDA also produced non-specific amplification artifacts when no DNA was used. RCA-RCA amplified DNA was generally of a high molecular weight (mostly above 10 kb), but with increasing storage of the FFPE specimen, the amplified fragment size reduced (0.3-4 kb in specimens stored for 29 months). Real-time PCR analysis revealed that RCA-RCA increased the amount of DNA by 600-4000 fold, but amplification amount depended on gene and storage duration. Generally, copy number profiles were similar in amplified and unamplifed DNA from FFPE, but when the DNA was highly degraded (stored 29 months), this correlation was lost. CGH profiles were similar in unamplified and RCA-RCA amplified DNA from FFPE specimens. In contrast, MDA amplified DNA was unsuitable for CGH.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Other
Platform:
Analyte Technology Platform DNA CGH DNA Real-time qPCR DNA Whole genome amplification Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Gliomas
Whole genome amplification Specific Nucleic acid amplification RCA-RCA
MDA
Storage Storage duration 17 months
20 months
29 months
Real-time qPCR Specific Targeted nucleic acid E2F
HBEGF
CYC
GAPDH
HER2
IL9R
TOP