{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,30]],"date-time":"2026-10-30T18:23:37Z","timestamp":1793384617808,"version":"4.5.0"},"reference-count":77,"publisher":"Public Library of Science (PLoS)","issue":"4","license":[{"start":{"date-parts":[[2022,4,26]],"date-time":"2022-04-26T00:00:00Z","timestamp":1650931200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002582","name":"Gwangju Institute of Science and Technology","doi-asserted-by":"publisher","award":["GIST Research Institute grant (2021)"],"award-info":[{"award-number":["GIST Research Institute grant (2021)"]}],"id":[{"id":"10.13039\/501100002582","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>The usage of cladocerans as non-model organisms in ecotoxicological and risk assessment studies has intensified in recent years due to their ecological importance in aquatic ecosystems. The molecular assessment such as gene expression analysis has been introduced in ecotoxicological and risk assessment to link the expression of specific genes to a biological process in the cladocerans. The validity and accuracy of gene expression analysis depends on the quantity, quality and integrity of extracted ribonucleic acid (RNA) of the sample. However, the standard methods of RNA extraction from the cladocerans are still lacking. This study evaluates the extraction of RNA from tropical freshwater cladocerans <jats:italic>Moina micrura<\/jats:italic> using two methods: the phenol-chloroform extraction method (QIAzol) and a column-based kit (Qiagen Micro Kit). Glycogen was introduced in both approaches to enhance the recovery of extracted RNA and the extracted RNA was characterised using spectrophotometric analysis (NanoDrop), capillary electrophoresis (Bioanalyzer). Then, the extracted RNA was analysed with reverse transcription polymerase chain reaction (RT-PCR) to validate the RNA extraction method towards downstream gene expression analysis. The results indicate that the column-based kit is most suitable for the extraction of RNA from <jats:italic>M<\/jats:italic>. <jats:italic>micrura<\/jats:italic>, with the quantity (RNA concentration = 26.90 \u00b1 6.89 ng\/\u03bcl), quality (A260:230 = 1.95 \u00b1 0.15, A280:230 = 1.85 \u00b1 0.09) and integrity (RNA integrity number, RIN = 7.20 \u00b1 0.16). The RT-PCR analysis shows that the method successfully amplified both alpha tubulin and actin gene at 33\u201335 cycles (i.e. Ct = 32.64 to 33.48). The results demonstrate that the addition of glycogen is only suitable for the phenol-chloroform extraction method. RNA extraction with high and comprehensive quality control assessment will increase the accuracy and reliability of downstream gene expression, thus providing more ecotoxicological data at the molecular biological level on other freshwater zooplankton species.<\/jats:p>","DOI":"10.1371\/journal.pone.0264989","type":"journal-article","created":{"date-parts":[[2022,4,26]],"date-time":"2022-04-26T17:52:32Z","timestamp":1650995552000},"page":"e0264989","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":7,"title":["Assessment of RNA extraction protocols from cladocerans"],"prefix":"10.1371","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7358-050X","authenticated-orcid":true,"given":"Muhammad Raznisyafiq","family":"Razak","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4827-0750","authenticated-orcid":true,"given":"Ahmad Zaharin","family":"Aris","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fatimah","family":"Md Yusoff","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zetty Norhana Balia","family":"Yusof","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sang Don","family":"Kim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kyoung Woong","family":"Kim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2022,4,26]]},"reference":[{"key":"pone.0264989.ref001","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1016\/j.tiv.2017.01.004","article-title":"Alternative approaches for identifying acute systemic toxicity: Moving from research to regulatory testing","volume":"41","author":"J Hamm","year":"2017","journal-title":"Toxicol Vitr"},{"key":"pone.0264989.ref002","doi-asserted-by":"crossref","first-page":"174","DOI":"10.1080\/10408444.2019.1579169","article-title":"Regulatory assessment and risk management of chemical mixtures: challenges and ways forward","volume":"49","author":"SK Bopp","year":"2019","journal-title":"Crit Rev Toxicol"},{"key":"pone.0264989.ref003","doi-asserted-by":"crossref","first-page":"437","DOI":"10.1016\/j.chemosphere.2017.01.101","article-title":"The fate of pharmaceuticals and personal care products (PPCPs), endocrine disrupting contaminants (EDCs), metabolites and illicit drugs in a WWTW and environmental waters","volume":"174","author":"E Archer","year":"2017","journal-title":"Chemosphere"},{"key":"pone.0264989.ref004","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1016\/j.jsps.2013.11.002","article-title":"Alternatives to animal testing: A review","volume":"23","author":"SK Doke","year":"2015","journal-title":"Saudi Pharm 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Pollut"},{"key":"pone.0264989.ref009","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/j.jes.2015.05.002","article-title":"Significance of adverse outcome pathways in biomarker-based environmental risk assessment in aquatic organisms","volume":"35","author":"JW Lee","year":"2015","journal-title":"J Environ Sci (China)"},{"key":"pone.0264989.ref010","doi-asserted-by":"crossref","first-page":"111905","DOI":"10.1016\/j.ecoenv.2021.111905","article-title":"Accumulation and risk assessment of heavy metals employing species sensitivity distributions in Linggi River, Negeri Sembilan, Malaysia","volume":"211","author":"MR Razak","year":"2021","journal-title":"Ecotoxicol Environ Saf"},{"key":"pone.0264989.ref011","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1016\/j.aquatox.2017.07.003","article-title":"Multigenerational effects evaluation of the flame retardant tris(2-butoxyethyl) phosphate (TBOEP) using Daphnia magna","volume":"190","author":"M Giraudo","year":"2017","journal-title":"Aquat Toxicol"},{"key":"pone.0264989.ref012","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1007\/s10646-018-2009-2","article-title":"Evaluation of 4-nonylphenol and bisphenol A toxicity using multiple molecular biomarkers in the water flea Daphnia magna","volume":"28","author":"RO Kim","year":"2019","journal-title":"Ecotoxicology"},{"key":"pone.0264989.ref013","doi-asserted-by":"crossref","first-page":"3850","DOI":"10.1021\/acs.est.8b06720","article-title":"Genome-Wide Stress Responses to Copper and Arsenic in a Field Population of Daphnia","volume":"53","author":"J Asselman","year":"2019","journal-title":"Environ Sci Technol"},{"key":"pone.0264989.ref014","doi-asserted-by":"crossref","first-page":"12898","DOI":"10.1021\/acs.est.7b05050","article-title":"Multigenerational Effects of the Antibiotic Tetracycline on Transcriptional Responses of Daphnia magna and Its Relationship to Higher Levels of Biological 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Liu","year":"2017","journal-title":"Aquat Toxicol"},{"key":"pone.0264989.ref018","doi-asserted-by":"crossref","first-page":"554","DOI":"10.3906\/zoo-1909-8","article-title":"Acute and chronic effects of thifluzamide on daphnia magna","volume":"43","author":"F. \u0130\u00e7o\u011flu Aksakal","year":"2019","journal-title":"Turkish J Zool"},{"key":"pone.0264989.ref019","doi-asserted-by":"crossref","first-page":"100008","DOI":"10.1016\/j.ese.2019.100008","article-title":"Zooplankton biodiversity monitoring in polluted freshwater ecosystems: A technical review","volume":"1","author":"W Xiong","year":"2020","journal-title":"Environ Sci Ecotechnology"},{"key":"pone.0264989.ref020","doi-asserted-by":"crossref","first-page":"282","DOI":"10.1016\/j.aquatox.2016.10.013","article-title":"Potential of the small cyclopoid copepod Paracyclopina nana as an invertebrate model for ecotoxicity testing","volume":"180","author":"H Dahms","year":"2016","journal-title":"Aquat Toxicol"},{"key":"pone.0264989.ref021","doi-asserted-by":"crossref","first-page":"2114","DOI":"10.1002\/etc.2665","article-title":"Acute toxicity tests and meta-analysis identify gaps in tropical ecotoxicology for amphibians","volume":"33","author":"SL Ghose","year":"2014","journal-title":"Environ Toxicol Chem"},{"key":"pone.0264989.ref022","first-page":"2018","article-title":"Comparison of Diazinon Toxicity to Temperate and Tropical Freshwater Daphnia Species","author":"TL Pham","year":"2018","journal-title":"J Chem"},{"key":"pone.0264989.ref023","doi-asserted-by":"crossref","first-page":"569","DOI":"10.11646\/zootaxa.4006.3.9","article-title":"Cladocera of Hainan Island, China","volume":"4006","author":"AY Sinev","year":"2015","journal-title":"Zootaxa"},{"key":"pone.0264989.ref024","doi-asserted-by":"crossref","first-page":"3","DOI":"10.4038\/sljas.v23i1.7542","article-title":"Zooplankton, zooplanktivorous fish and their interactions in Southeast Asian waterbodies with special reference to Sri Lanka: a review","volume":"23","author":"J Vijverberg","year":"2018","journal-title":"Sri Lanka J Aquat Sci"},{"key":"pone.0264989.ref025","first-page":"1210","article-title":"Culture of the zooplankton as fish food: Observations on three freshwater species from Assam, India","volume":"10","author":"S Kar","year":"2017","journal-title":"AACL Bioflux"},{"key":"pone.0264989.ref026","doi-asserted-by":"crossref","DOI":"10.5772\/18275","article-title":"Using Zooplankton, Moina Micrura Kurz to Evaluate the Ecotoxicology of Pesticides Used in Paddy Fields of Thailand","author":"C Boonthai","year":"2011","journal-title":"Pestic Mod World\u2014Risks Benefits"},{"key":"pone.0264989.ref027","doi-asserted-by":"crossref","first-page":"518","DOI":"10.1590\/1519-6984.14113","article-title":"On the first record of the exotic Moina macrocopa (Straus, 1820) in Minas Gerais State, Brazil","volume":"74","author":"AC Rietzler","year":"2014","journal-title":"Brazilian J Biol"},{"key":"pone.0264989.ref028","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1134\/S207511171802011X","article-title":"The Distribution of Moina micrura Kurz, 1875 (Crustacea: Moinidae) in the Russian Part of the Vistula Lagoon (Baltic Sea)","volume":"9","author":"AS Semenova","year":"2018","journal-title":"Russ J Biol Invasions"},{"key":"pone.0264989.ref029","doi-asserted-by":"crossref","first-page":"227","DOI":"10.23818\/limn.38.18","article-title":"Who is Moina micrura? Redescription of one of the most confusing cladocerans from terra typica, based on integrative taxonomy","volume":"38","author":"M El\u00edas-Guti\u00e9rrez","year":"2019","journal-title":"Limnetica"},{"key":"pone.0264989.ref030","first-page":"229","article-title":"Acute and Chronic Effects of Three Pharmaceutical Drugs on the Tropical Freshwater Cladoceran Ceriodaphnia silvestrii","author":"LL Damasceno de Oliveira","year":"2018","journal-title":"Water Air Soil Pollut"},{"key":"pone.0264989.ref031","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1080\/21553769.2016.1162753","article-title":"Bioindicators: the natural indicator of environmental pollution","volume":"9","author":"TK Parmar","year":"2016","journal-title":"Front Life Sci"},{"key":"pone.0264989.ref032","doi-asserted-by":"crossref","first-page":"2689","DOI":"10.1002\/etc.3819","article-title":"Single and combined effects of microcystin- and saxitoxin-producing cyanobacteria on the fitness and antioxidant defenses of cladocerans","volume":"36","author":"A da S Ferr\u00e3o-Filho","year":"2017","journal-title":"Environ Toxicol Chem"},{"key":"pone.0264989.ref033","doi-asserted-by":"crossref","DOI":"10.5772\/18275","article-title":"Using Zooplankton, Moina Micrura Kurz to Evaluate the Ecotoxicology of Pesticides Used in Paddy Fields of Thailand","author":"C Boonthai","year":"2011","journal-title":"Pesticides in the Modern World\u2014Risks and Benefits"},{"key":"pone.0264989.ref034","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1016\/j.aquaculture.2013.06.034","article-title":"Effect of food density on male appearance and ephippia production in a tropical cladoceran, Moina micrura Kurz, 1874","volume":"412\u2013413","author":"OM Azuraidi","year":"2013","journal-title":"Aquaculture"},{"key":"pone.0264989.ref035","first-page":"1","article-title":"Four Transgenerational Demographic Performance of Moina macrocopa Exposed to Chronic Levels of Cadmium","volume":"15","author":"GJL Flores","year":"2017","journal-title":"Dose-Response"},{"key":"pone.0264989.ref036","doi-asserted-by":"crossref","first-page":"445","DOI":"10.1007\/s10646-007-0148-y","article-title":"A simple and rapid method for preserving RNA of aquatic invertebrates for ecotoxicogenomics","volume":"16","author":"LH Heckmann","year":"2007","journal-title":"Ecotoxicology"},{"key":"pone.0264989.ref037","doi-asserted-by":"crossref","first-page":"393","DOI":"10.1093\/plankt\/fbz025","article-title":"Sex determination and gene expression in Daphnia magna exposed to juvenile hormone","volume":"41","author":"M Wuerz","year":"2019","journal-title":"J Plankton Res"},{"key":"pone.0264989.ref038","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1002\/lom3.10079","article-title":"Ethanol: A simple and effective RNA-preservation for freshwater insects living in remote habitats","volume":"14","author":"T Astrid","year":"2016","journal-title":"Limnol Oceanogr Methods"},{"key":"pone.0264989.ref039","doi-asserted-by":"crossref","first-page":"599","DOI":"10.1016\/j.mex.2018.05.011","article-title":"Optimization of phenol-chloroform RNA extraction","volume":"5","author":"LS Toni","year":"2018","journal-title":"MethodsX"},{"key":"pone.0264989.ref040","first-page":"1","article-title":"Optimisation of laboratory methods for whole transcriptomic RNA analyses in human left ventricular biopsies and blood samples of clinical relevance","volume":"14","author":"KL Ford","year":"2019","journal-title":"PLoS One"},{"key":"pone.0264989.ref041","doi-asserted-by":"crossref","first-page":"6397","DOI":"10.1007\/s00216-017-0580-7","article-title":"An improvement of miRNA extraction efficiency in human plasma","volume":"409","author":"E Ban","year":"2017","journal-title":"Anal Bioanal Chem"},{"key":"pone.0264989.ref042","first-page":"1","article-title":"Total RNA extraction from tissues for microRNA and target gene expression analysis: Not all kits are created equal","volume":"18","author":"RAM Brown","year":"2018","journal-title":"BMC Biotechnol"},{"key":"pone.0264989.ref043","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1093\/biomethods\/bpy012","article-title":"RNA profile diversity across arthropoda: Guidelines, methodological artifacts, and expected outcomes","volume":"3","author":"DM Deleo","year":"2018","journal-title":"Biol Methods Protoc"},{"key":"pone.0264989.ref044","doi-asserted-by":"crossref","first-page":"650","DOI":"10.1111\/j.1755-0998.2011.03010.x","article-title":"Next-generation sequencing of transcriptomes: A guide to RNA isolation in nonmodel animals","volume":"11","author":"P Gayral","year":"2011","journal-title":"Mol Ecol Resour"},{"key":"pone.0264989.ref045","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1016\/j.cca.2016.09.018","article-title":"Optimization of total RNA isolation from human urinary sediment","volume":"462","author":"MB Monteiro","year":"2016","journal-title":"Clin Chim Acta"},{"key":"pone.0264989.ref046","doi-asserted-by":"crossref","first-page":"100006","DOI":"10.1016\/j.xpro.2019.100006","article-title":"Generation of Inexpensive, Highly Labeled Probes for Fluorescence In Situ Hybridization (FISH)","volume":"1","author":"R Sharma","year":"2020","journal-title":"STAR Protoc"},{"key":"pone.0264989.ref047","doi-asserted-by":"crossref","DOI":"10.1007\/978-3-642-17890-0","volume-title":"Guidelines for Molecular Analysis in Archive Tissues","author":"G. Stanta","year":"2011"},{"key":"pone.0264989.ref048","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.bdq.2018.02.001","article-title":"A PCR-based quantitative assay for the evaluation of mRNA integrity in rat samples","volume":"15","author":"BK Padhi","year":"2018","journal-title":"Biomol Detect Quantif"},{"key":"pone.0264989.ref049","unstructured":"Krupp G. Stringent RNA quality control using the Agilent 2100 bioanalyzer. 2005. Available: http:\/\/www.chem.agilent.com\/Library\/applications\/5989-1086EN.pdf."},{"key":"pone.0264989.ref050","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1371\/journal.pone.0187590","article-title":"Do microplastic particles affect Daphnia magna at the morphological, life history and molecular level?","volume":"12","author":"HK Imhof","year":"2017","journal-title":"PLoS One"},{"key":"pone.0264989.ref051","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/j.margen.2014.12.004","article-title":"Marine Genomics High-quality RNA extraction from copepods for Next Generation Sequencing: A comparative study","volume":"24","author":"S Asai","year":"2015","journal-title":"Mar Genomics"},{"key":"pone.0264989.ref052","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3800\/pbr.11.1","article-title":"Ontogenetic variations in dry mass and nucleic acid contents of Artemia salina","volume":"11","author":"T Kobari","year":"2016","journal-title":"Plankt Benthos Res"},{"key":"pone.0264989.ref053","first-page":"119","article-title":"qPCR detection versus microscopy observations for assessing presence\u2013absence of Didymosphenia geminata in Quebec rivers (Canada)","volume":"52","author":"S Kim Tiam","year":"2017","journal-title":"Water Qual Res J Canada"},{"key":"pone.0264989.ref054","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1016\/j.jembe.2013.04.021","article-title":"An improved method for achieving high-quality RNA for copepod transcriptomic studies","volume":"446","author":"H Zhang","year":"2013","journal-title":"J Exp Mar Bio Ecol"},{"key":"pone.0264989.ref055","unstructured":"Macedo NJ, Ferreira TL. Maximizing Total RNA Yield from TRIzol\u00ae Reagent Protocol: A Feasibility Study. ASEE 2014 Zone I Conference. 2014."},{"key":"pone.0264989.ref056","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1673\/031.010.14119","article-title":"Why does insect RNA look degraded?","volume":"10","author":"EC Winnebeck","year":"2010","journal-title":"J Insect Sci"},{"key":"pone.0264989.ref057","first-page":"11","article-title":"A combined rna preservation and extraction protocol for gene expression studies in cacao beans","author":"DJ Wever","year":"2020","journal-title":"Front Plant Sci"},{"key":"pone.0264989.ref058","doi-asserted-by":"crossref","first-page":"1684","DOI":"10.1261\/rna.046011.114","article-title":"Power analysis and sample size estimation for RNA-Seq differential expression","volume":"20","author":"T Ching","year":"2014","journal-title":"Rna"},{"key":"pone.0264989.ref059","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1016\/j.mam.2005.12.003","article-title":"RNA integrity and the effect on the real-time qRT-PCR performance","volume":"27","author":"S Fleige","year":"2006","journal-title":"Mol Aspects Med"},{"key":"pone.0264989.ref060","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1089\/bio.2017.0099","article-title":"Effect of Postmortem Interval and Years in Storage on RNA Quality of Tissue at a Repository of the NIH NeuroBioBank","volume":"16","author":"K White","year":"2018","journal-title":"Biopreserv Biobank"},{"key":"pone.0264989.ref061","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1007\/s13530-017-0302-8","article-title":"Acute toxicity and antioxidant responses in the water flea Daphnia magna to xenobiotics (cadmium, lead, mercury, bisphenol A, and 4-nonylphenol)","volume":"9","author":"H Kim","year":"2017","journal-title":"Toxicol Environ Health Sci"},{"key":"pone.0264989.ref062","doi-asserted-by":"crossref","first-page":"884","DOI":"10.1111\/1755-0998.12122","article-title":"Optimizing preservation protocols to extract high-quality RNA from different tissues of echinoderms for next-generation sequencing","volume":"13","author":"P-R Portela","year":"2013","journal-title":"Mol Ecol Resour"},{"key":"pone.0264989.ref063","doi-asserted-by":"crossref","first-page":"532","DOI":"10.1089\/bio.2012.0030","article-title":"Short-term stability study of RNA at room temperature","volume":"10","author":"C Mathay","year":"2012","journal-title":"Biopreserv Biobank"},{"key":"pone.0264989.ref064","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1371\/journal.pone.0121659","article-title":"RNA preservation agents and nucleic acid extraction method bias perceived bacterial community composition","volume":"10","author":"A McCarthy","year":"2015","journal-title":"PLoS One"},{"key":"pone.0264989.ref065","doi-asserted-by":"crossref","first-page":"662","DOI":"10.1111\/1755-0998.12485","article-title":"Evaluation of the impact of RNA preservation methods of spiders for de novo transcriptome assembly","volume":"16","author":"N Kono","year":"2016","journal-title":"Mol Ecol Resour"},{"key":"pone.0264989.ref066","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1371\/journal.pone.0172171","article-title":"High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos","volume":"12","author":"N Ruocco","year":"2017","journal-title":"PLoS One"},{"key":"pone.0264989.ref067","doi-asserted-by":"crossref","first-page":"143","DOI":"10.4319\/lom.2005.3.143","article-title":"Effects of preservation and storage of microcrustaceans in RNAlater on RNA and DNA degradation","volume":"3","author":"E. Gorokhova","year":"2005","journal-title":"Limnol Oceanogr Methods"},{"key":"pone.0264989.ref068","doi-asserted-by":"crossref","first-page":"972","DOI":"10.1093\/plankt\/fbt035","article-title":"Acetone preservation for zooplankton molecular studies","volume":"35","author":"E Goetze","year":"2013","journal-title":"J Plankton Res"},{"key":"pone.0264989.ref069","article-title":"A low-cost and easy-to-use cell preservation reagent for 4\u00b0C or room temperature sample storage","author":"S Shankar","year":"2019","journal-title":"bioRxiv"},{"key":"pone.0264989.ref070","doi-asserted-by":"crossref","first-page":"45","DOI":"10.4319\/lom.2011.1.45","article-title":"Ethanol: a better preservation technique for Daphnia","volume":"1","author":"AR Black","year":"2011","journal-title":"Limnol Oceanogr Methods"},{"key":"pone.0264989.ref071","doi-asserted-by":"crossref","first-page":"386","DOI":"10.1002\/elps.201900200","article-title":"Highly degraded RNA can still provide molecular information: An in vitro approach","volume":"41","author":"P Fattorini","year":"2020","journal-title":"Electrophoresis"},{"key":"pone.0264989.ref072","first-page":"1","article-title":"A survey of best practices for RNA-seq data analysis","volume":"17","author":"A Conesa","year":"2016","journal-title":"Genome Biol"},{"key":"pone.0264989.ref073","doi-asserted-by":"crossref","first-page":"3298","DOI":"10.1096\/fj.201601031RR","article-title":"Effects of RNA integrity on transcript quantification by total RNA sequencing of clinically collected human placental samples","volume":"31","author":"M Reiman","year":"2017","journal-title":"FASEB J"},{"key":"pone.0264989.ref074","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s13059-019-1682-7","article-title":"DegNorm: Normalization of generalized transcript degradation improves accuracy in RNA-seq analysis","volume":"20","author":"B Xiong","year":"2019","journal-title":"Genome Biol"},{"key":"pone.0264989.ref075","doi-asserted-by":"crossref","first-page":"730","DOI":"10.1002\/etc.34","article-title":"Adverse outcome pathways: A conceptual framework to support ecotoxicology research and risk assessment","volume":"29","author":"GT Ankley","year":"2010","journal-title":"Environ Toxicol Chem"},{"key":"pone.0264989.ref076","doi-asserted-by":"crossref","first-page":"971","DOI":"10.1002\/jat.3165","article-title":"Adverse outcome pathways can drive non-animal approaches for safety assessment","volume":"35","author":"N Burden","year":"2015","journal-title":"J Appl Toxicol"},{"key":"pone.0264989.ref077","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1093\/toxsci\/kfv181","article-title":"Adverse outcome pathways for regulatory applications: Examination of four case studies with different degrees of completeness and scientific confidence","volume":"148","author":"EJ Perkins","year":"2015","journal-title":"Toxicol Sci"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0264989","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,4,26]],"date-time":"2022-04-26T17:54:08Z","timestamp":1650995648000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0264989"}},"subtitle":[],"editor":[{"given":"SSS","family":"Sarma","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2022,4,26]]},"references-count":77,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2022,4,26]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0264989","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,4,26]]}}}