DNA degradation test predicts success in whole-genome amplification from diverse clinical samples.
Author(s): Wang F, Wang L, Briggs C, Sicinska E, Gaston SM, Mamon H, Kulke MH, Zamponi R, Loda M, Maher E, Ogino S, Fuchs CS, Li J, Hader C, Makrigiorgos GM
Publication: J Mol Diagn, 2007, Vol. 9, Page 441-51
PubMed ID: 17690213 PubMed Review Paper? No
Suggested by: ISBER
Purpose of Paper
The purpose of this paper was to compare methods to predict real-time PCR success after restriction and circularization-aided rolling circle amplification (RCA-RCA) whole genome amplification (WGA) of DNA from stored formalin-fixed and paraffin-embedded (FFPE) glioblastomas and colorectal carcinomas and from circulating plasma DNA.
Conclusion of Paper
DNA gliomas/glioblastomas stored for 5-7 years generally yielded more intact DNA than the colorectal carcinomas stored for 10-12 years. DNA degradation, as detected by electrophoresis, did not correspond to real-time PCR success after RCA-RCA amplification, but multiplex PCR success, evaluated with denaturing high-performance liquid chromatography (dHPLC) instead of electrophoresis, was a better indicator of subsequent real-time PCR success after RCA-RCA amplification.
Studies
-
Study Purpose
The purpose of this study was to determine the effects of storage and specimen type on DNA integrity and real-time PCR success after RCA-RCA WGA. DNA was extracted from snap-frozen and xylene-deparaffinized, proteinase K-digested FFPE specimens using DNEasy. Plasma DNA was obtained using QIAamp MinElute.
Summary of Findings:
DNA from 13 of the 16 gliomas stored for 5-7 years was intact as determined by electrophoresis and multiplex PCR. The remaining 3 specimens showed weakened amplification of products longer than 100 bp. After excluding the 3 specimens that showed degradation, all gliomas stored 5-7 years allowed for real-time PCR amplification after whole genome RCA-RCA amplification of at least 18 of the 20 genes tested. DNA from the colorectal carcinomas stored for 10-12 years was generally degraded, and only 1 of the 12 specimens allowed for equal amplification of all 4 multiplex PCR products. After RCA-RCA amplification, 2 of the 12 colorectal specimens allowed for real-time PCR amplification of all 20 genes. The remaining 10 RCA-RCA amplified colorectal specimens allowed for real-time PCR amplification of 4-18 of the 20 genes. Importantly, DNA degradation, as determined by electrophoresis, did not correspond to real-time PCR success after RCA-RCA amplification, but multiplex PCR success, evaluated with dHPLC instead of electrophoresis, was a better, but still not perfect, indicator of subsequent real-time PCR success after RCA-RCA amplification. Multiplex PCR dHPLC was able to predict subsequent real-time PCR success after RCA-RCA amplification from plasma-circulating DNA.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Other
Platform:
Analyte Technology Platform DNA Electrophoresis DNA Whole genome amplification DNA PCR DNA HPLC DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 5-7 years
10-12 years
Biospecimen Acquisition Biospecimen location Glioma
Glioblastoma
Colorectal
Plasma
Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Snap frozen
PCR Specific Technology platform Electrophoresis
Real-time PCR
PCR Specific Length of gene fragment 105 bp
236 bp
299 bp
411 bp
PCR Specific Targeted nucleic acid GAPDH
PCR Specific Detection method Electrophoresis with ethidium bromide
dHPLC