The effects of tissue fixation on sequencing and transcript abundance of nucleic acids from microdissected liver samples of smallmouth bass (Micropterus dolomieu).

Autor: Walsh HL; U.S. Geological Survey, National Fish Health Research Laboratory, Leetown Science Center, Kearneysville, West Virginia, United States of America., Sperry AJ; U.S. Geological Survey, National Fish Health Research Laboratory, Leetown Science Center, Kearneysville, West Virginia, United States of America., Blazer VS; U.S. Geological Survey, National Fish Health Research Laboratory, Leetown Science Center, Kearneysville, West Virginia, United States of America.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2020 Aug 10; Vol. 15 (8), pp. e0236104. Date of Electronic Publication: 2020 Aug 10 (Print Publication: 2020).
DOI: 10.1371/journal.pone.0236104
Abstrakt: There is an increasing emphasis on effects-based monitoring to document responses associated with exposure to complex mixtures of chemicals, climate change, pathogens, parasites and other environmental stressors in fish populations. For decades aquatic monitoring programs have included the collection of tissues preserved for microscopic pathology. Consequently, formalin-fixed, paraffin-embedded (FFPE) tissue can be an important reservoir of nucleic acids as technologies emerge that utilize molecular endpoints. Despite the cross-linking effects of formalin, its impact on nucleic acid quality and concentration, amplification, and sequencing are not well described. While fresh-frozen tissue is optimal for working with nucleic acids, FFPE samples have been shown to be conducive for molecular studies. Laser capture microdissection (LCM) is one technology which allows for collection of specific regions or cell populations from fresh or preserved specimens with pathological alterations, pathogens, or parasites. In this study, smallmouth bass (Micropterus dolomieu) liver was preserved in three different fixatives, including 10% neutral buffered formalin (NBF), Z-Fix® (ZF), and PAXgene® (PG) for four time periods (24 hr, 48 hr, seven days, and 14 days). Controls consisted of pieces of liver preserved in RNALater® or 95% ethanol. Smallmouth bass were chosen as they are an economically important sportfish and have been utilized as indicators of exposure to endocrine disruptors and other environmental stressors. Small liver sections were cut out with laser microdissection and DNA and RNA were purified and analyzed for nucleic acid concentration and quality. Sanger sequencing and the NanoString nCounter® technology were used to assess the suitability of these samples in downstream molecular techniques. The results revealed that of the formalin fixatives, NBF samples fixed for 24 and 48 hr were superior to ZF samples for both Sanger sequencing and the Nanostring nCounter®. The non-formalin PAXgene® samples were equally successful and they showed greater stability in nucleic acid quality and concentration over longer fixation times. This study demonstrated that small quantities of preserved tissue from smallmouth bass can be utilized in downstream molecular techniques; however, future studies will need to optimize the methods presented here for different tissue types, fish species, and pathological conditions.
Competing Interests: The authors have declared that no competing interests exist.
Databáze: MEDLINE
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