The influence of fixation temperature in in vitro DNA analysis

Canhoto, I.; Duarte, C.; Rego, B.; Marques-Ramos, Ana

Publication

The influence of fixation temperature in in vitro DNA analysis

2018-01Conference object

dc.contributor.author	Canhoto, I.
dc.contributor.author	Duarte, C.
dc.contributor.author	Rego, B.
dc.contributor.author	Marques-Ramos, Ana
dc.date.accessioned	2018-06-12T11:57:46Z
dc.date.available	2018-06-12T11:57:46Z
dc.date.issued	2018-01
dc.description.abstract	Introduction: Formalin-fixed and paraffin-embedded (FFPE) are important sources for molecular studies, namely to identify cancer related biomarkers. Nevertheless, the quality of FFPE-obtained DNA is lower than that of fresh/frozen tissues, since fixation induces several chemical modifications. The impact of tissue fixation duration, fixative type and pH on the integrity of FFPE-extracted DNA has been the subject of several studies. It’s well established that fixation using formalin for less than 72 hours allows extraction of high quality DNA. Although it is known that DNA stability is highly dependent on temperature, the influence of the fixation temperature on the quality of FFPE-extracted DNA is not understood. Objective: Evaluate the influence of the fixation temperature in the quality of FFPE-extracted DNA through a systematic literature review. Materials and methods: The search was performed in PubMed for studies published in English, up to December 2017. The included studies compared two or more fixation temperatures using formalin, to perform DNA analysis. Results: Seven studies met the defined criteria. All compared room temperature (RT) with 4 °C (5 studies), 0-4 °C (1 study) or 37 °C (1 study). DNA integrity was evaluated by agarose gel electrophoresis electrophoresis, PCR, multiplex ligation-dependent probe amplification (MLPA) and whole gene amplification (WGA). In 5 studies the best results were obtained for fixation at 4 °C (electrophoresis, PCR and WGA); whereas RT allowed the best results in 2 cases (PCR and MLPA), one of which demonstrated that fixation at 37 °C preserved DNA similarly to RT. Conclusions: DNA stability is highly temperature-dependent as DNAses are inhibited at low temperatures. Accordingly, it is not surprising that fixation at 4 °C allows the extraction of less degraded DNA in most studies. Nevertheless, RT seems to be also an acceptable temperature, as it allowed successful MLPA and PCR when using small DNA fragments. From these observations one may conclude that tissue fixation must be performed at 4 °C if the goal is to analyse high-molecular-weight DNA. As RT is the standard fixation temperature in diagnostic lab units, these conclusions may imply the adequate adjustments in order to prevent false conclusions from molecular analysis. Nevertheless, additional studies that analyse not only DNA integrity, but also DNA purity, yield and concentration should be done to determine the optimal fixation temperature to perform molecular analysis.	pt_PT
dc.description.version	info:eu-repo/semantics/publishedVersion	pt_PT
dc.identifier.citation	Canhoto I, Duarte C, Rego B, Marques-Ramos A. The influence of fixation temperature in in vitro DNA analysis. In: CIMAGO Scientific Meeting, Faculty of Medicine, University of Coimbra (Portugal), January 25-26, 2018. Poster 43. Pulmonol. 2018;24(Esp Cong 1):20.	pt_PT
dc.identifier.uri	http://hdl.handle.net/10400.21/8625
dc.language.iso	eng	pt_PT
dc.peerreviewed	yes	pt_PT
dc.publisher	Elsevier	pt_PT
dc.relation.publisherversion	http://meeting.acimago.com/	pt_PT
dc.rights.uri	http://creativecommons.org/licenses/by-nc-nd/4.0/	pt_PT
dc.subject	FFPE	pt_PT
dc.subject	Formalin-fixed and paraffin-embedded	pt_PT
dc.subject	In vitro DNA	pt_PT
dc.subject	Fixation temperature	pt_PT
dc.title	The influence of fixation temperature in in vitro DNA analysis	pt_PT
dc.type	conference object
dspace.entity.type	Publication
oaire.citation.endPage	20	pt_PT
oaire.citation.startPage	20	pt_PT
oaire.citation.title	Pulmonology	pt_PT
oaire.citation.volume	24	pt_PT
person.familyName	Marques-Ramos
person.givenName	Ana
person.identifier.ciencia-id	0617-3550-881E
person.identifier.orcid	0000-0003-2655-8806
rcaap.rights	openAccess	pt_PT
rcaap.type	conferenceObject	pt_PT
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