{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,16]],"date-time":"2026-10-16T22:35:31Z","timestamp":1792190131600,"version":"4.4.0"},"reference-count":33,"publisher":"Public Library of Science (PLoS)","issue":"3","license":[{"start":{"date-parts":[[2023,3,29]],"date-time":"2023-03-29T00:00:00Z","timestamp":1680048000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100009708","name":"Novo Nordisk Fonden","doi-asserted-by":"publisher","award":["31364"],"award-info":[{"award-number":["31364"]}],"id":[{"id":"10.13039\/501100009708","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>The use of fresh tissue for molecular studies is preferred but often impossible. Instead, frozen or formalin-fixed, paraffin-embedded (FFPE) tissues are widely used and constitute valuable resources for retrospective studies. We assessed the utility of cardiac tissue stored in different ways for gene expression analyses by whole transcriptome sequencing of paired fresh, frozen, and FFPE tissues. RNA extracted from FFPE was highly degraded. Sequencing of RNA from FFPE tissues yielded higher proportions of intronic and intergenic reads compared to RNA from fresh and frozen tissues. The global gene expression profiles varied with the storage conditions, particularly mitochondrial and long non-coding RNAs. However, we observed high correlations among protein-coding transcripts (\u03c1 &gt; 0.94) with the various storage conditions. We did not observe any significant storage effect on the allele-specific gene expression. However, FFPE had statistically significantly (p &lt; 0.05) more discordant variant calls compared to fresh and frozen tissue. In conclusion, we found that frozen and FFPE tissues can be used for reliable gene expression analyses, provided that proper quality control is performed and caution regarding the technical variability is withheld.<\/jats:p>","DOI":"10.1371\/journal.pone.0283159","type":"journal-article","created":{"date-parts":[[2023,3,29]],"date-time":"2023-03-29T17:51:55Z","timestamp":1680112315000},"page":"e0283159","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":64,"title":["Comparison of whole transcriptome sequencing of fresh, frozen, and formalin-fixed, paraffin-embedded cardiac tissue"],"prefix":"10.1371","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6884-2701","authenticated-orcid":true,"given":"Stine B\u00f8ttcher","family":"Jacobsen","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jacob","family":"Tfelt-Hansen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Morten Holdgaard","family":"Smerup","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeppe Dyrberg","family":"Andersen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9463-5087","authenticated-orcid":true,"given":"Niels","family":"Morling","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2023,3,29]]},"reference":[{"key":"pone.0283159.ref001","article-title":"Differentially expressed genes for atrial fibrillation identified by rna sequencing from paired human left and right atrial appendages","author":"AM Thomas","year":"2019","journal-title":"Physiol Genomics"},{"key":"pone.0283159.ref002","article-title":"RNA-Seq identifies novel myocardial gene expression signatures of heart failure","author":"Y Liu","year":"2015","journal-title":"Genomics"},{"key":"pone.0283159.ref003","article-title":"Myocardial infarction biomarker discovery with integrated gene expression, pathways and biological networks analysis","author":"A Mujalli","year":"2020","journal-title":"Genomics"},{"key":"pone.0283159.ref004","article-title":"Identification of Atrial Fibrillation-Associated Genes ERBB2 and MYPN Using Genome-Wide Association and Transcriptome Expression Profile Data on Left\u2013Right Atrial Appendages.","author":"X Meng","year":"2021","journal-title":"Front Genet."},{"key":"pone.0283159.ref005","article-title":"Analysis of long non-coding RNA and mRNA profiles in epicardial adipose tissue of patients with atrial fibrillation","author":"L Zhao","year":"2020","journal-title":"Biomed Pharmacother"},{"key":"pone.0283159.ref006","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1007\/978-1-4939-8935-5_22","article-title":"Making Formalin-Fixed, Paraffin Embedded Blocks.","volume":"1897","author":"A Sadeghipour","year":"2019","journal-title":"Methods Mol Biol"},{"key":"pone.0283159.ref007","article-title":"Why Formalin-fixed, Paraffin-embedded Biospecimens Must Be Used in Genomic Medicine: An Evidence-based Review and Conclusion","author":"W Mathieson","year":"2020","journal-title":"J Histochem Cytochem"},{"key":"pone.0283159.ref008","doi-asserted-by":"crossref","first-page":"2361","DOI":"10.1002\/elps.200600589","article-title":"Multiplex PCR with minisequencing as an effective high-throughput SNP typing method for formalin-fixed tissue","volume":"28","author":"MTP Gilbert","year":"2007","journal-title":"Electrophoresis"},{"key":"pone.0283159.ref009","article-title":"Sequence artifacts in DNA from formalin-fixed tissues: Causes and strategies for minimization","author":"H Do","year":"2015","journal-title":"Clinical Chemistry"},{"key":"pone.0283159.ref010","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1371\/journal.pone.0170632","article-title":"A comparison of RNA-Seq results from paired formalin-fixed paraffin-embedded and fresh-frozen glioblastoma tissue samples.","volume":"12","author":"A Esteve-Codina","year":"2017","journal-title":"PLoS One"},{"key":"pone.0283159.ref011","doi-asserted-by":"crossref","first-page":"6980","DOI":"10.1038\/s41598-019-43282-8","article-title":"Deleterious effects of formalin-fixation and delays to fixation on RNA and miRNA-Seq profiles","volume":"9","author":"W Jones","year":"2019","journal-title":"Sci Rep"},{"key":"pone.0283159.ref012","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0098187","article-title":"Next-generation sequencing of RNA and DNA isolated from paired fresh-frozen and formalin-fixed paraffin-embedded samples of human cancer and normal tissue","volume":"9","author":"J Hedegaard","year":"2014","journal-title":"PLoS One"},{"key":"pone.0283159.ref013","first-page":"1","article-title":"Robustness of RNA sequencing on older formalin-fixed paraffin-embedded tissue from high-grade ovarian serous adenocarcinomas","volume":"14","author":"Y Zhao","year":"2019","journal-title":"PLoS One"},{"key":"pone.0283159.ref014","doi-asserted-by":"crossref","first-page":"695","DOI":"10.1136\/jclinpath-2017-204969","article-title":"Evaluation of the quality of RNA extracted from archival FFPE glioblastoma and epilepsy surgical samples for gene expression assays","volume":"71","author":"HR Haynes","year":"2018","journal-title":"J Clin Pathol"},{"key":"pone.0283159.ref015","article-title":"Large scale, robust, and accurate whole transcriptome profiling from clinical formalin-fixed paraffin-embedded samples.","author":"Y Newton","year":"2020","journal-title":"Sci Rep."},{"key":"pone.0283159.ref016","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41598-020-71521-w","article-title":"Direct formalin fixation induces widespread transcriptomic effects in archival tissue samples.","volume":"10","author":"LC Wehmas","year":"2020","journal-title":"Sci Rep"},{"key":"pone.0283159.ref017","article-title":"FastQC\u2014A quality control tool for high throughput sequence data","author":"S. Andrews","year":"2010","journal-title":"In: Babraham Bioinformatics"},{"key":"pone.0283159.ref018","article-title":"AdapterRemoval: Easy cleaning of next-generation sequencing reads.","author":"S. Lindgreen","year":"2012","journal-title":"BMC Res Notes."},{"key":"pone.0283159.ref019","article-title":"STAR: Ultrafast universal RNA-seq aligner","author":"A Dobin","year":"2013","journal-title":"Bioinformatics"},{"key":"pone.0283159.ref020","article-title":"GENCODE reference annotation for the human and mouse genomes","author":"A Frankish","year":"2019","journal-title":"Nucleic Acids Res"},{"key":"pone.0283159.ref021","doi-asserted-by":"crossref","DOI":"10.1007\/978-3-319-24277-4","volume-title":"ggplot2: Elegant Graphics for Data Analysis","author":"H. Wickham","year":"2016"},{"key":"pone.0283159.ref022","volume-title":"R: A language and environment for statistical computing","author":"R Core Team","year":"2020"},{"key":"pone.0283159.ref023","article-title":"edgeR: A Bioconductor package for differential expression analysis of digital gene expression data","author":"MD Robinson","year":"2009","journal-title":"Bioinformatics"},{"key":"pone.0283159.ref024","doi-asserted-by":"crossref","first-page":"e47","DOI":"10.1093\/nar\/gkv007","article-title":"Limma powers differential expression analyses for RNA-sequencing and microarray studies","volume":"43","author":"ME Ritchie","year":"2015","journal-title":"Nucleic Acids Res"},{"key":"pone.0283159.ref025","doi-asserted-by":"crossref","first-page":"1381","DOI":"10.1038\/s41436-021-01172-3","article-title":"ACMG SF v3.0 list for reporting of secondary findings in clinical exome and genome sequencing: a policy statement of the American College of Medical Genetics and Genomics (ACMG).","volume":"23","author":"DT Miller","year":"2021","journal-title":"Genet Med"},{"key":"pone.0283159.ref026","first-page":"1307","article-title":"European Heart Rhythm Association (EHRA)\/Heart Rhythm Society (HRS)\/Asia Pacific Heart Rhythm Society (APHRS)\/Latin American Heart Rhythm Society (LAHRS) Expert Consensus Statement on the state of genetic testing for cardiac diseases.","volume":"24","author":"AAM Wilde","year":"2022","journal-title":"EP Eur"},{"key":"pone.0283159.ref027","volume-title":"RStudio: Integrated Development for R.","author":"RStudio T","year":"2020"},{"key":"pone.0283159.ref028","article-title":"Comparison of triple-negative breast cancer molecular subtyping using RNA from matched fresh-frozen versus formalin-fixed paraffin-embedded tissue","author":"B Jovanovi\u0107","year":"2017","journal-title":"BMC Cancer"},{"key":"pone.0283159.ref029","article-title":"Robust gene expression and mutation analyses of RNA-sequencing of formalin-fixed diagnostic tumor samples","author":"S Graw","year":"2015","journal-title":"Sci Rep"},{"key":"pone.0283159.ref030","article-title":"Whole genome and transcriptome sequencing of post-mortem cardiac tissues from sudden cardiac death victims identifies a gene regulatory variant in NEXN","author":"JD Andersen","year":"2019","journal-title":"Int J Legal Med"},{"key":"pone.0283159.ref031","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s12864-018-4761-3","article-title":"mRNA-seq whole transcriptome profiling of fresh frozen versus archived fixed tissues","volume":"19","author":"N Bossel Ben-Moshe","year":"2018","journal-title":"BMC Genomics"},{"key":"pone.0283159.ref032","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1371\/journal.pone.0203608","article-title":"Impact of storage conditions on the quality of nucleic acids in paraffin embedded tissues.","volume":"13","author":"D Groelz","year":"2018","journal-title":"PLoS One."},{"key":"pone.0283159.ref033","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1007\/s00428-017-2213-0","article-title":"DNA degrades during storage in formalin-fixed and paraffin-embedded tissue blocks","volume":"471","author":"A Guyard","year":"2017","journal-title":"Virchows Arch"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0283159","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,3,29]],"date-time":"2023-03-29T17:52:48Z","timestamp":1680112368000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0283159"}},"subtitle":[],"editor":[{"given":"Alessandro","family":"Weisz","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2023,3,29]]},"references-count":33,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2023,3,29]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0283159","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,3,29]]}}}