Pathobiology : journal of immunopathology, molecular and cellular biology · 2022
Tissue Fixation with a Formic Acid-Deprived Formalin Better Preserves DNA Integrity over Time.
СтатьяНазвание и аннотация: Перевод в очереди
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Berrino Enrico, Annaratone Laura, Detillo Paolo, Grassini Dora, Bragoni Alberto, Sapino Anna, Bussolati Benedetta, Bussolati Giovanni, Marchiò Caterina
Аннотация
INTRODUCTION: Optimization of pre-analytic procedures and tissue processing is a basic requirement for reliable and reproducible data to be obtained. Tissue fixation in formalin represents the extensively favored method for surgical tissue specimen processing in diagnostic pathology; however, formalin fixation exerts a blasting effect on DNA and RNA.
METHODS: A formic acid-deprived formaldehyde solution was prepared by removing acids with an ion-exchange basic resin and the concentrated, acid-deprived formaldehyde (ADF) solution was employed to prepare a 4% ADF solution in 0.1 M phosphate buffer, pH 7.2-7.4. Human (n = 27) and mouse (n = 20) tissues were fixed in parallel and similar conditions in either ADF or neutral buffered formalin (NBF). DNAs and RNAs were extracted, and fragmentation analyses were performed.
RESULTS: Besides no significant differences in terms of extraction yield and absorbance ratio, ADF fixation reduced DNA fragmentation, i.e., the largest fragments (>5,000 bp) were significantly more prevalent in the DNAs purified from ADF-fixed tissues (p < 0.001 in both cohorts). Moreover, we observed that DNA preservation is more stable in ADF-fixed tissue compared to NBF-fixed tissues.
CONCLUSION: Although DNA fragmentation in FFPE tissues is a multifactor process, we showed that the removal of formic acid is responsible for a significant improvement in DNA preservation.
METHODS: A formic acid-deprived formaldehyde solution was prepared by removing acids with an ion-exchange basic resin and the concentrated, acid-deprived formaldehyde (ADF) solution was employed to prepare a 4% ADF solution in 0.1 M phosphate buffer, pH 7.2-7.4. Human (n = 27) and mouse (n = 20) tissues were fixed in parallel and similar conditions in either ADF or neutral buffered formalin (NBF). DNAs and RNAs were extracted, and fragmentation analyses were performed.
RESULTS: Besides no significant differences in terms of extraction yield and absorbance ratio, ADF fixation reduced DNA fragmentation, i.e., the largest fragments (>5,000 bp) were significantly more prevalent in the DNAs purified from ADF-fixed tissues (p < 0.001 in both cohorts). Moreover, we observed that DNA preservation is more stable in ADF-fixed tissue compared to NBF-fixed tissues.
CONCLUSION: Although DNA fragmentation in FFPE tissues is a multifactor process, we showed that the removal of formic acid is responsible for a significant improvement in DNA preservation.