Published online Sep 24, 2019. doi: 10.5306/wjco.v10.i9.307
Peer-review started: March 20, 2019
First decision: April 15, 2019
Revised: August 19, 2019
Accepted: September 4, 2019
Article in press: September 5, 2019
Published online: September 24, 2019
Processing time: 203 Days and 9.3 Hours
Nucleic acid isolation from formalin-fixed, paraffin-embedded tissue (FFPET) samples is a daily routine in molecular pathology laboratories, but extraction from FFPET is not always easily achieved. Choosing the right extraction technique is key for further examinations. Several commercial kits are available on the molecular biology market, including both manual isolation procedures and automated extraction. When choosing the right method for isolation, consideration must be given to the aspects of time, precision, downstream applications and price. Choosing the right technique is key for success in molecular biology, because nucleic acid isolation is always the first step in molecular biology and molecular pathology.
The aim of this paper was to compare the advantages and disadvantages of four DNA extraction kits used in daily practice for DNA isolation from formalin-fixed, paraffin-embedded (FFPE) colorectal adenocarcinoma (CRC) surgical specimens: two manual and two automated magnetic bead kits. A correlation of the results with the clinicopathological features of CRCs was also performed.
By comparing the advantages and disadvantages of nucleic acid isolation techniques used in daily routines, precise decisions can be made regarding the most suitable DNA extraction approach for molecular applications.
DNA was extracted from FFPE-CRCs. The selection of tumor area was based on the presence of tumor cells in over 80% of the marked tissue, without necroses, hemorrhages, inflammatory, or highly fibrotic stroma. For manual DNA isolation, two commercially available kits were used: The PureLink Genomic DNA Mini Kit from Invitrogen Carlsbad, CA 92008, United States and the High Pure FFPE DNA Isolation Kit from Roche GmbH, Mannheim, Germany. For automated DNA isolation, two commercially available automated magnetic bead kits were used: The iPrep Genomic DNA Kit from Invitrogen and the MagnaPure LC DNA Isolation Kit from Roche. DNA parameters (concentration and quality) were determined using a Nanodrop machine (ThermoScientific, United States). Readings were taken at wavelengths of 260 nm and 280 nm. The optical density (OD) ratio (A260/A280) was automatically calculated, before being correlated with tumor localization, macroscopic and microscopic features, the depth of infiltration, the lymph node ratio, and tumor stage, which were determined according to the latest classification rules.
DNA concentration was influenced by the macroscopic features and grade of differentiation. A higher DNA concentration was obtained for polypoid compared with ulcero-infiltrative carcinomas, with both Roche systems and using the automated system from Invitrogen. The manual kit from Invitrogen allowed good concentrations to be extracted, but in half of the cases (23 of 46 cases) a value below 150 ng/µL was obtained. For this reason, the P value was considered to be at the limit of statistical significance.
Manual methods of DNA extraction are more controllable and allow the in-house adaptation of the protocol. The obtained DNA concentrations and purity are higher. Automated methods are a time-saving option for polymerase chain reaction (PCR) and real-time quantitative PCR reactions. For CRC samples, a higher DNA concentration is expected to be obtained from differentiated polypoid carcinomas.
DNA integrity is higher when manual purification is performed, for both tissues and whole blood. The unresolved issue refers to the imbalance between concentration and quality. The above-mentioned aspects should be investigated in larger cohorts with sample size calculations.