{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T01:52:37Z","timestamp":1781661157281,"version":"3.54.5"},"reference-count":26,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2021,10,24]],"date-time":"2021-10-24T00:00:00Z","timestamp":1635033600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2021,10,24]],"date-time":"2021-10-24T00:00:00Z","timestamp":1635033600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["BMC Genomics"],"published-print":{"date-parts":[[2021,12]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec>\n                <jats:title>Background<\/jats:title>\n                <jats:p>Archival formalin-fixed, paraffin-embedded (FFPE) tissue samples with clinical and histological data are a singularly valuable resource for developing new molecular biomarkers. However, transcriptome analysis remains challenging with standard mRNA-seq methods as FFPE derived-RNA samples are often highly modified and fragmented. The recently developed 3\u2032 mRNA-seq method sequences the 3\u2032 region of mRNA using unique molecular identifiers (UMI), thus generating gene expression data with minimal PCR bias. In this study, we evaluated the performance of 3\u2032 mRNA-Seq using Lexogen QuantSeq 3\u2032 mRNA-Seq Library Prep Kit FWD with UMI, comparing with TruSeq Stranded mRNA-Seq and RNA Exome Capture kit. The fresh-frozen (FF) and FFPE tissues yielded nucleotide sizes range from 13 to &gt;\u200970% of DV200 values; input amounts ranged from 1\u2009ng to 100\u2009ng for validation.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>The total mapped reads of QuantSeq 3\u2032 mRNA-Seq to the reference genome ranged from 99 to 74% across all samples. After PCR bias correction, 3 to 56% of total sequenced reads were retained. QuantSeq 3\u2032 mRNA-Seq data showed highly reproducible data across replicates in Universal Human Reference RNA (UHR, R\u2009&gt;\u20090.94) at input amounts from 1\u2009ng to 100\u2009ng, and FF and FFPE paired samples (R\u2009=\u20090.92) at 10\u2009ng. Severely degraded FFPE RNA with \u226430% of DV200 value showed good concordance (R\u2009&gt;\u20090.87) with 100\u2009ng input. A moderate correlation was observed when directly comparing QuantSeq 3\u2032 mRNA-Seq data with TruSeq Stranded mRNA-Seq (R\u2009=\u20090.78) and RNA Exome Capture data (R\u2009&gt;\u20090.67).<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusion<\/jats:title>\n                <jats:p>In this study, QuantSeq 3\u2032 mRNA-Seq with PCR bias correction using UMI is shown to be a suitable method for gene quantification in both FF and FFPE RNAs. 3\u2032 mRNA-Seq with UMI may be applied to severely degraded RNA from FFPE tissues generating high-quality sequencing data.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s12864-021-08068-1","type":"journal-article","created":{"date-parts":[[2021,10,24]],"date-time":"2021-10-24T11:02:25Z","timestamp":1635073345000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Application of the 3\u2032 mRNA-Seq using unique molecular identifiers in highly degraded RNA derived from formalin-fixed, paraffin-embedded tissue"],"prefix":"10.1186","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7199-4418","authenticated-orcid":false,"given":"Jin Sung","family":"Jang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eileen","family":"Holicky","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julie","family":"Lau","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Samantha","family":"McDonough","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark","family":"Mutawe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew J.","family":"Koster","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kenneth J.","family":"Warrington","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julie M.","family":"Cuninngham","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,10,24]]},"reference":[{"issue":"1","key":"8068_CR1","doi-asserted-by":"publisher","first-page":"125","DOI":"10.1016\/j.ccell.2017.12.004","volume":"33","author":"R Yaeger","year":"2018","unstructured":"Yaeger R, Chatila WK, Lipsyc MD, Hechtman JF, Cercek A, Sanchez-Vega F, et al. 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As required by Mayo Clinic Institutional Review Board policies, all subjects provided written research consent or authorization for the use of their tissues and data.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not Applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"None.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"759"}}