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. 2017 Jan 1;147(1):60-68.
doi: 10.1093/ajcp/aqw205.

Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA

Lilit Atanesyan  1 , Maryvonne J Steenkamer  1 , Anja Horstman  2 , Cathy B Moelans  3 , Jan P Schouten  1 , Suvi P Savola  1
Affiliations

Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA

Lilit Atanesyan et al. Am J Clin Pathol. .

Abstract

Objectives: Molecular genetic analysis of formalin-fixed, paraffin-embedded (FFPE) tissues is of great importance both for research and diagnostics. Multiplex ligation-dependent probe amplification (MLPA) is a widely used technique for gene copy number determination, and it has been successfully used for FFPE tissue-extracted DNA analysis. However, there have been no studies addressing the effect of tissue fixation procedures and DNA extraction methods on MLPA. This study therefore focuses on selecting optimal preanalytic conditions such as FFPE tissue preparation conditions and DNA extraction methods.

Methods: Healthy tissues were fixed in buffered or nonbuffered formalin for 1 hour, 12 to 24 hours, or 48 to 60 hours at 4 °C or at room temperature. DNA extracted from differently fixed and subsequently paraffin-embedded tissues was used for MLPA. Four commercial DNA extraction kits and one in-house method were compared.

Results: Tissues fixed for 12 to 24 hours in buffered formalin at room temperature produced DNA with the most optimal quality for MLPA. The in-house FFPE DNA extraction method was shown to perform as efficient as or even superior to other methods in terms of suitability for MLPA, time and cost-efficiency, and ease of performance.

Conclusions: FFPE-extracted DNA is well suitable for MLPA analysis, given that optimal tissue fixation and DNA extraction methods are chosen.

Keywords: Copy number; DNA extraction; FFPE; Formalin fixation; MLPA; Multiplex ligation-dependent probe amplification.

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Figures

Figure 1
Figure 1
Multiplex ligation-dependent probe amplification (MLPA) probe copy number ratio deviation as a result of different formalin-fixed, paraffin-embedded tissue fixation conditions. Two MLPA probe mixes are presented in separate columns: P027 (white) and P105 (gray). Tissue fixation conditions are indicated on the x-axis. The percentage of probes with copy number ratios that fall outside the normal copy number ratio range (0.8-1.2) is indicated on the y-axis. RT, room temperature.
Figure 2
Figure 2
The influence of five different DNA extraction methods on the multiplex ligation-dependent probe amplification (MLPA) probe’s copy number ratios. MLPA was performed on DNA extracted from eight various formalin-fixed, paraffin-embedded (FFPE) tissues with the P027 probe mix (50 probes). The y-axis represents the percentage of probes showing copy number ratios outside the 0.8 to 1.2 normal copy number range. The x-axis shows five different DNA extraction methods. Each bar represents a separate FFPE tissue-type block. For samples where DNA was extracted with the one-tube FFPE extraction method, results from crude (nonpurified) DNA lysate are presented for all tissues, except the lung. The crude lysate of the lung FFPE tissue was also purified. SEM (as indicated by error bars) was calculated by dividing the standard deviation of the mean of the number of probes with copy number ratios outside the normal range by the square root of the number of samples (triplicate) (Supplementary Table S6). colonSymb, tissue from Symbiant Pathology Expert Centre, Alkmaar; colonUMC, tissue from Department of Pathology, University Medical Centre Utrecht, Utrecht.
Figure 3
Figure 3
(cont) C, MLPA electropherogram of FFPE lung tissue crude lysate column purified with the DNA Clean & Concentrator-5 kit. RFU, residual fluorescence unit.
Figure 3
Figure 3
(cont) C, MLPA electropherogram of FFPE lung tissue crude lysate column purified with the DNA Clean & Concentrator-5 kit. RFU, residual fluorescence unit.
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