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

Author(s): Atanesyan L, Steenkamer MJ, Horstman A, Moelans CB, Schouten JP, Savola SP

Publication: Am J Clin Pathol, 2017, Vol. , Page

PubMed ID: 28122725 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated how DNA copy number ratios may be affected by the formula of formalin used (buffered versus unbuffered formalin) and the duration and temperature of fixation in formalin-fixed paraffin-embedded (FFPE) colon specimens. Three 4 mm thick sections of a healthy colon specimen were processed with a Leica TP1050 and fixed in 10% formalin for 1 h, 12-24 h or 48-60 h at room temperature; in 10% formalin for 12-24 h or 48-60 h at 4˚C; or in unbuffered formalin (pH 3.0) for 12-24 h at room temperature. DNA was extracted from three 10 µm thick sections of 1 cm² paraffin blocks using an unspecified method. Multiple ligation-dependent probe amplification (MLPA) was conducted using two probes sets (consisting of 50 and 55 probes, respectively) and a standard MLPA one-tube protocol and commercially-obtained DNA from the blood of one male or female donor as a copy number reference.

   Summary of Findings:

   Copy number ratios for all probes were within the normal range of 0.8 and 1.2 when specimens were fixed for 12-24 h at room temperature. The percentage of probes that fell outside of the normal range for copy number ratio was 2-4% when specimens were fixed in buffered formalin at room temperature for 1 h, and 14-18% when specimens were fixed for 48-60 h. Similarly, 4-5% of probes fell outside of the normal range for copy number ratio when specimens were fixed in buffered formalin at 4˚C for 12-24 h, while 70-75% of probes were outside of the normal range when specimens were fixed in unbuffered formalin at room temperature for 12-24 h.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Formalin

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Time in fixative	1 h 12-24 h 48-60 h
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Formalin (unbuffered)
   Biospecimen Preservation	Temperature of fixation/preservation	4˚C Room temperature

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2. Study Purpose

   This study investigated whether DNA extraction method influences the percentage of probes that was within the normal range for copy number ratio (0.8 and 1.2) when FFPE specimens are anlyzed. A single tissue specimen was collected from eight healthy donors and fixed in 10% buffered formalin at room temperature for 12-24 h. DNA was extracted from three 10 µm thick sections of kidney, prostate, stomach, colon, brain, bone marrow and lung, and ten 10 µm sections of breast. Five different DNA extraction methods were evaluated: (1) the QIAamp DNA FFPE tissue kit with overnight lysis at 56˚C, (2) the RecoverAll Total Nucleic Acid Isolation kit for FFPE which included overnight lysis at 50˚C, (3) the ZR FFPE DNA MiniPrep, (4) the WaxFree DNA extraction Kit for Paraffin Samples, and (5) an in-house protocol that included deparaffinization via a 90˚C incubation in extraction buffer, overnight Proteinase K digestion at 55˚C,  incubation at 80˚C for 15 min, and centrifugation. For lung, copy number ratio results were also compared with and without column based purification od extraction DNA with a DNA Clean & Concentrator-5 Kit. DNA yield was determined by Q-ratio and purity by spectrophotometry. MLPA was conducted using two probes sets (consisting of 50 and 55 probes, respectively) and a standard MLPA one-tube protocol using commercially-obtained DNA from the blood of one male or female donor as the copy number reference.

   Summary of Findings:

   The in-house, one-tube DNA extraction method generated the largest percentage of probes (73-99%) that fell within the normal range for copy number ratio (0.8-1.2), followed by the ZR FFPE DNA MiniPrep kit (59-99%), the WaxFree DNA extraction Kit (29-99%), the QIAamp DNA extraction kit (29-91%), and the RecoverAll Total Nucleic Acid Isolation Kit (38-68%). Importantly, copy number ratio reproducibility was also highest when DNA was extracted using the in-house method.  Tissue type also influenced results, as the highest percentages of probes within the normal range for copy number ratio was observed for kidney and the lowest for brain and lung. For lung tissue specimens, the percentage of probes that fell within the normal range for copy number ratio was only 30-40% when DNA was extracted using the WaxFree and in-house methods; however, column based purification post-extraction increased that percentage to approximately 80%. Electropherograms revealed the presence of a large amount of unused primer when DNA was amplified without purification, but the residual primer peak fell below the average peak height when samples were purified. 

   Biospecimens

   • Tissue - Bone Marrow
   • Tissue - Brain
   • Tissue - Colorectal
   • Tissue - Stomach
   • Tissue - Lung
   • Tissue - Prostate
   • Tissue - Kidney

   Preservative Types

   • Formalin

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	QIAamp DNA FFPE tissue kit RecoverAll Total Nucleic Acid Isolation kit for FFPE ZR FFPE DNA MiniPrep WaxFree DNA extraction Kit for Paraffin Samples In-house heating protocol
   Biospecimen Acquisition	Biospecimen location	Colon Bone marrow Lung Kidney Prostate Stomach Brain
   Analyte Extraction and Purification	Protein digestion	Overnight at 56˚C Overnight at 50˚C
   Analyte Extraction and Purification	Deparaffinization	Xylene Heating to 90˚C in extraction buffer

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