Observational Study
Copyright ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Clin Oncol. Sep 24, 2019; 10(9): 307-317
Published online Sep 24, 2019. doi: 10.5306/wjco.v10.i9.307
DNA extraction from paraffin embedded colorectal carcinoma samples: A comparison study of manual vs automated methods, using four commercially kits
Zsolt Kovacs, Ioan Jung, Erzsebet Csernak, Zoltan Szentirmay, Laura Banias, Genoveva Rigmanyi, Simona Gurzu
Zsolt Kovacs, Ioan Jung, Laura Banias, Simona Gurzu, Department of Pathology, University of Medicine, Pharmacy, Sciences and Technology, Targu-Mures 530149, Romania
Genoveva Rigmanyi, Simona Gurzu, Research Center (CCAMF), University of Medicine, Pharmacy, Sciences and Technology, Targu-Mures 540139, Romania
Author contributions: Kovacs Z drafted the article and contributed to the DNA purifications; Jung I contributed to the diagnosis and immunohistochemical assessment; Csernak E contributed to automated and manual DNA isolation; Szentirmay Z contributed to the research design; Banias Laura performed DNA collection; Rigmanyi G performed manual DNA extraction; Gurzu S designed research and confer the final agreement for publication; Zsolt Kovacs and Laura Banias have equally contribution to the paper.
Supported by the University of Medicine, Pharmacy, Science and Technology Research Grant, No. 275/11.01.2017.
Institutional review board statement: The agreement of the Ethical Committee of University of Medicine and Pharmacy, Targu-Mures, Romania, was obtained.
Informed consent statement: This is a retrospective study. No consent was necessary.
Conflict-of-interest statement: The authors have declared no conflicts of interest.
Data sharing statement: No additional data are available.
STROBE statement: The authors have read the STROBE Statement-checklist of items, and the manuscript was prepared and revised according to the STROBE statement-checklist of items.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Simona Gurzu, MD, PhD, Chief Doctor, Research Center (CCAMF), University of Medicine, Pharmacy, Sciences and Technology, 38 Gheorghe Marinescu Street, Targu-Mures 540139, Romania. simonagurzu@yahoo.com
Telephone: +40-745-673550 Fax: +40-265-210407
Received: March 12, 2019
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
ARTICLE HIGHLIGHTS
Research background

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.

Research motivation

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.

Research objectives

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.

Research methods

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.

Research results

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.

Research conclusions

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.

Research perspectives

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.