Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue.
Author(s): Potluri K, Mahas A, Kent MN, Naik S, Markey M
Publication: Anal Biochem, 2015, Vol. 486, Page 17-23
PubMed ID: 26126956 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare DNA yield, maximum PCR amplicon size and array-based comparative genomic hybridization (aCGH) success in formalin-fixed paraffin-embedded (FFPE) skin biopsies extracted using three different DNA extraction methods.
Conclusion of Paper
While DNA yield was greatest when specimens were extracted with the QIAamp kit, extraction using the adaptive-focused acoustics (AFA)-based truXTRAC kit generated longer maximum PCR amplicons, which extended genotype determination by aCGH to include longer restriction fragments compared to other extractions methods (600 bp versus 400-500 bp). DNA yields determined by NanoDrop were 2.7-6.6-fold higher than when determined by Qubit, although the magnitude of difference was influenced by extraction method.
Studies
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Study Purpose
The purpose of this study was to determine the effect of DNA extraction method on DNA yield, maximum PCR amplicon size and aCGH success using FFPE skin biopsies. DNA was extracted using each of the three methods from 27 benign skin biopsies that had been stored 8 or 11 years. Prior to DNA extraction using the QIAamp FFPE kit or standard phenol-chloroform, 24 10µm sections were deparaffinization with xylene and proteinase K digested for 72 h. DNA was extracted from 8-10 10 µm sections using the AFA-based Covaris truXTRAC FFPE DNA kit which uses an ultrasonicator for deparaffinization and homogenization before a 2 h proteinase K digestion. When aCGH success was compared, DNA was extracted from 54 skin biopsies using the QIAamp kit and from 9 biopsies using the truXTRAC kit.
Summary of Findings:
DNA yield was 2.7-6.6-fold higher when quantified by NanoDrop than by Qubit. The largest discrepancy was observed among specimens extracted using phenol chloroform and the smallest among those extracted using the QIAamp kit. Significantly higher DNA yields per section (as determined by Qubit) were obtained using the QIAamp kit compared to either the truXTRAC kit (p=0.000079) or phenol chloroform (p=0.0000947), and yield was slightly higher using truXTRAC than phenol-chloroform (p=0.033). The maximum amplicon size was comparable between phenol chloroform and QIAamp extracted specimens (346.7 and 347.4 bp, respectively), but was larger (401.9 bp) for truXTRAC extracted specimens (p=0.04 and p=0.03, respectively). Importantly, DNA extraction by truXTRAC permitted aCGH genotype determination in fragments as long as 600 bp, but the maximum fragment lengths that generated aCGH data with QIAamp extracted specimens were 400-500 bp, depending on enzyme used for digestion.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Benign
Platform:
Analyte Technology Platform DNA Fluorometry DNA Electrophoresis DNA Spectrophotometry DNA Array CGH Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Qiagen QIAamp DNA FFPE tissue kit
Phenol–chloroform extraction
Covaris truXTRAC FFPE DNA kit
Spectrophotometry Specific Technology platform NanoDrop
Qubit
Array CGH Specific Template modification Nsp1 digested
Sty1 digested