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Currently, several RNA-Seq methods have been developed. However, the relative advantage of each method for degraded RNA and low-input RNA, such as RNA samples collected in the field of clinical setting, has remained unknown. The Standard method of RNA-Seq captures mRNA by poly(A) capturing using Oligo dT beads, which is not suitable for degraded RNA. Here, we used three commercially available RNA-Seq library preparation kits (SMART-Seq, xGen Broad-range, and RamDA-Seq) using random primer instead of Oligo dT beads. To evaluate the performance of these methods, we compared the correlation, the number of detected expressing genes, and the expression levels with the Standard RNA-Seq method. Although the performance of RamDA-Seq was similar to that of Standard RNA-Seq, the performance for low-input RNA and degraded RNA has decreased. The performance of SMART-Seq was better than xGen and RamDA-Seq in low-input RNA and degraded RNA. Furthermore, the depletion of ribosomal RNA (rRNA) improved the performance of SMART-Seq and xGen due to increased expression levels. SMART-Seq with rRNA depletion has relative advantages for RNA-Seq using low-input and degraded RNA.<\/jats:p>","DOI":"10.3390\/ijms25116143","type":"journal-article","created":{"date-parts":[[2024,6,3]],"date-time":"2024-06-03T10:06:01Z","timestamp":1717409161000},"page":"6143","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Comparison of RNA-Sequencing Methods for Degraded RNA"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7600-3481","authenticated-orcid":false,"given":"Hiroki","family":"Ura","sequence":"first","affiliation":[{"name":"Center for Clinical Genomics, Kanazawa Medical University Hospital, 1-1 Daigaku, Uchinada, Kahoku 920-0923, Japan"},{"name":"Division of Genomic Medicine, Department of Advanced Medicine, Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahoku 920-0923, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5641-7962","authenticated-orcid":false,"given":"Yo","family":"Niida","sequence":"additional","affiliation":[{"name":"Center for Clinical Genomics, Kanazawa Medical University Hospital, 1-1 Daigaku, Uchinada, Kahoku 920-0923, Japan"},{"name":"Division of Genomic Medicine, Department of Advanced Medicine, Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahoku 920-0923, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,6,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1038\/nmeth.1226","article-title":"Mapping and quantifying mammalian transcriptomes by RNA-Seq","volume":"5","author":"Mortazavi","year":"2008","journal-title":"Nat. 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