{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,6]],"date-time":"2026-03-06T08:06:23Z","timestamp":1772784383085,"version":"3.50.1"},"reference-count":33,"publisher":"Public Library of Science (PLoS)","issue":"12","license":[{"start":{"date-parts":[[2024,12,26]],"date-time":"2024-12-26T00:00:00Z","timestamp":1735171200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100007048","name":"Mayo Foundation for Medical Education and Research","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100007048","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Archived FFPE cardiac tissue specimens are valuable for molecular studies aimed at identifying biomarkers linked to mortality in cardiovascular disease. Establishing a reliable and reproducible RNA extraction method is critical for generating high-quality transcriptome sequences for molecular assays. Here, the efficiency of four RNA extraction methods: Qiagen AllPrep DNA\/RNA method (Method QP); Qiagen AllPrep DNA\/RNA method with protocol modification on the ethanol wash step after deparaffinization (Method QE); CELLDATA RNA extraction (Method BP) and CELLDATA RNA extraction with protocol modifications on the lysis step (Method BL) was compared on 23 matching FFPE cardiac tissue specimens (n = 92).In comparing RNA quality metrics across FFPE RNA extract, nucleic acids extracted deploying Method QE and QP produced the highest RNA yield. However, Method QE outperformed Method QP as more extract from Method QE had DV 200 values above 30%. Both method BL and BP produced similar range of RNA purity and yield but more extract from Method BL had DV 200 values above 30% compared to Method BP. When accessing distribution value, Method BL outperformed Methods BP, QE, and QP as more extracts from Method BL had DV 200 values above 30% compared to other methods (P<jats:sub>DV200<\/jats:sub>&lt;0.001; Kruskal-Wallis). Method QE outperformed other methods in terms of RNA yield. RNA extracts from Method QE, characterized by high RNA yield, achieved sequencing results comparable to those from Method BL, characterized by high DV200 values. Our findings reveal that optimizing protocols can yield higher-quality RNA, facilitating the exploration of more disease conditions with high-resolution transcriptome profiling.<\/jats:p>","DOI":"10.1371\/journal.pone.0315098","type":"journal-article","created":{"date-parts":[[2024,12,26]],"date-time":"2024-12-26T18:26:15Z","timestamp":1735237575000},"page":"e0315098","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":2,"title":["Optimizing RNA extraction methods for high-throughput transcriptome sequencing of formalin-fixed paraffin-embedded cardiac tissue specimens"],"prefix":"10.1371","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7824-0096","authenticated-orcid":true,"given":"Nkechi Martina","family":"Odogwu","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jin Sung","family":"Jang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sabrina","family":"Albertson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Clinton","family":"Hagen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Boyd","family":"Rasmussen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Oommen","family":"Saji","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Timothy J.","family":"Nelson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"name":"the Wanek Program Preclinical Pipeline","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2024,12,26]]},"reference":[{"issue":"8","key":"pone.0315098.ref001","first-page":"e93","article-title":"Heart disease and stroke statistics\u20142023 update: a report from the American Heart Association","volume":"147","author":"C. 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Xing","year":"2006","journal-title":"Nucleic acids research"},{"issue":"12","key":"pone.0315098.ref021","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1186\/s13059-014-0550-8","article-title":"Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2","volume":"15","author":"M. I. Love","year":"2014","journal-title":"Genome biology"},{"issue":"1","key":"pone.0315098.ref022","first-page":"btac830","article-title":"igv.js: an embeddable JavaScript implementation of the Integrative Genomics Viewer (IGV)","volume":"39","author":"J. T. Robinson","year":"2023","journal-title":"Bioinformatics (Oxford, England)"},{"key":"pone.0315098.ref023","unstructured":"R Core Team (2018). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https:\/\/www.R-project.org\/"},{"issue":"18","key":"pone.0315098.ref024","doi-asserted-by":"crossref","first-page":"e144","DOI":"10.1093\/nar\/gks588","article-title":"Quantification of RNA integrity and its use for measurement of transcript number","volume":"40","author":"M. J. Brisco","year":"2012","journal-title":"Nucleic acids research"},{"issue":"6","key":"pone.0315098.ref025","doi-asserted-by":"crossref","first-page":"e0179732","DOI":"10.1371\/journal.pone.0179732","article-title":"Reliability and performance of commercial RNA and DNA extraction kits for FFPE tissue cores","volume":"12","author":"P. G. Patel","year":"2017","journal-title":"PloS one"},{"key":"pone.0315098.ref026","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1186\/1471-2199-7-3","article-title":"The RIN: an RNA integrity number for assigning integrity values to RNA measurements","volume":"7","author":"A Schroeder","year":"2006","journal-title":"BMC Mol Biol"},{"issue":"7","key":"pone.0315098.ref027","doi-asserted-by":"crossref","first-page":"e70714","DOI":"10.1371\/journal.pone.0070714","article-title":"Quality control of RNA preservation and extraction from paraffin-embedded tissue: implications for RT-PCR and microarray analysis","volume":"8","author":"K. Kashofer","year":"2013","journal-title":"PLoS One"},{"key":"pone.0315098.ref028","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s12874-018-0628-1","article-title":"Quantity and quality of nucleic acids extracted from archival formalin fixed paraffin embedded prostate biopsies","volume":"18","author":"J. Carlsson","year":"2018","journal-title":"BMC Medical Research Methodology"},{"key":"pone.0315098.ref029","unstructured":"Illumina, R. N. A. (2016). Evaluating RNA quality from FFPE Samples. Technical Note: Library Preparation"},{"key":"pone.0315098.ref030","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1016\/j.atg.2016.06.001","article-title":"Standardization and quality management in next-generation sequencing","volume":"10","author":"C. Endrullat","year":"2016","journal-title":"Applied & translational genomics"},{"issue":"2\u20133","key":"pone.0315098.ref031","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. 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