Comparison of eight commercially available kits for DNA extraction from formalin-fixed paraffin-embedded tissues.
Author(s): Janecka A, Adamczyk A, Gasińska A
Publication: Anal Biochem, 2015, Vol. 476, Page 8-10
PubMed ID: 25640584 PubMed Review Paper? No
Purpose of Paper
This paper compared DNA yield, purity, and amplification among specimens isolated using eight different DNA extraction kits and investigated the effects of digestion duration and multiple elution steps.
Conclusion of Paper
The highest yields of DNA of acceptable purity were obtained using the QIAamp DNA FFPE Tissue and ReliaPrep FFPE gDNA kits. In most cases, increasing the digestion time from 3 h to overnight resulted in higher yields and some improvement in purity but had little to no effect on DNA obtained using the QIAamp DNA FFPE Tissue. Importantly, the first elution step for all kits contained the majority of the DNA and this DNA was significantly less contaminated by RNA/proteins or chemicals for six of eight kits.
Studies
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Study Purpose
This study investigated the effects of digestion duration, extraction kit, and number of elution steps on the DNA yield, purity, and ability to be amplified. DNA was extracted from ten normal kidney specimens from patients with renal cancer using eight different kits. For each kit, DNA was extracted from two 10µm sections according to the kit protocols with the exception of the use of xylene instead of octane in conjunction with the Invisorb Spin Tissue Mini Kit. Specimens were digested for 3 h or overnight and DNA was RNase treated and subsequently stored at -20˚C.
Summary of Findings:
When digestion was for 3 h, the highest DNA yield was obtained with the MagMAX FFPE DNA Isolation kit followed by the ZR FFPE DNA MiniPrep and QIAamp DNA FFPE Tissue kits, but the purity based on spectrophotometry was significantly lower using the Syngen Tissue DNA Mini, MagMAX FFPE DNA Isolation, GeneMATRIX Tissue DNA Purification, and E.Z.N.A. FFPE DNA kits than the QIAamp DNA FFPE Tissue kit. The authors conclude that only the QIAamp DNA FFPE Tissue kit allowed for isolation of pure DNA from specimens digested for 3 h. When digested for 3 h, only DNA isolated using the QIAamp DNA FFPE Tissue or ReliaPrep FFPE gDNA Miniprep kits, and to a lesser extent the Invisorb Spin Tissue Mini kit showed more amplification of PI3KCA than the internal control. Longer digestion resulted in higher DNA yields for six of the eight kits (lower yields for QIAamp DNA FFPE Tissue and MagMAX FFPE DNA Isolation kits). Purity as determined by A260/A280 and A260/A230 ratios were significantly increased for specimens extracted with the Syngen Tissue DNA Mini kit; A260/280 ratios were significantly increased when digestion was extended to overnight in conjunction with QIAamp DNA FFPE Tissue, MagMAX FFPE DNA Isolation, or GeneMATRIX Tissue DNA Purification kits; and A260/230 ratios were significantly increased in specimens extracted with E.Z.N.A. FFPE DNA or Invisorb Spin Tissue Mini kits when digestion was lengthened to overnight. Interestingly, a slight decline in A260/280 ratios were observed when specimens extracted with ZR FFPE DNA MiniPrep were digested overnight and a decline in A260/230 was noted when specimens extracted with kit MagMAX FFPE DNA Isolation Kit were digested overnight. The first elution step contained 58% (Invisorb Spin Tissue Mini) to 78% (MagMAX FFPE DNA Isolation kit) of the DNA and the A260/280 and A260/230 ratios were significantly higher for the first elution than the second for six of eight kits.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Normal Adjacent
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Spectrophotometry DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Syngen Tissue DNA Mini Kit
ZR FFPE DNA MiniPrep kit
QIAamp DNA FFPE Tissue kit
MagMAX FFPE DNA Isolation kit
GeneMATRIX Tissue DNA Purification kit
ReliaPrep FFPE gDNA Miniprep System
E.Z.N.A. FFPE DNA
Invisorb Spin Tissue Mini kit
Real-time qPCR Specific Targeted nucleic acid PIK3CA gene
Analyte Extraction and Purification Rehydration of dried sample/specimen Once
Twice
Analyte Extraction and Purification Protein digestion 3 h
Overnight