DV200 Index for assessing RNA integrity in next‐generation sequencing
Open accessAbstract
Poor quality of biological samples will result in an inaccurate analysis of next‐generation sequencing (NGS). Therefore, methods to accurately evaluate sample integrity are needed. Among methods for evaluating RNA quality, the RNA integrity number equivalent (RINe) is widely used, whereas the DV200, which evaluates the percentage of fragments of >200 nucleotides, is also used as a quality assessment standard. In this study, we compared the RINe and DV200 RNA quality indexes to determine the most suitable RNA index for the NGS analysis. Seventy‐one RNA samples were extracted from formalin‐fixed paraffin‐embedded tissue samples ( n = 30), fresh‐frozen samples ( n = 25), or cell lines ( n = 16). After assessing RNA quality using the RINe and DV200, we prepared two kinds of stranded mRNA sequencing libraries. Finally, we calculated the correlation between each RNA quality index and the amount of library product (1 st PCR product per input RNA). The DV200 measure showed stronger correlation with the amount of library product than the RINe ( R 2 = 0.8208 for the DV200 versus 0.6927 for the RINe). Receiver operating characteristic curve analyses revealed that the DV200 was the better marker for predicting efficient library production than the RINe using a threshold of >10 ng/ng for the amount of the 1 st PCR product per input RNA (cutoff value for the RINe and DV200, 2.3 and 66.1%; area under the curve, 0.99 and 0.91; sensitivity, 82% and 92%; and specificity, 93% and 100%, respectively). Our results indicate that NGS libraries prepared using RNA samples with the DV200 value > 66.1 % exhibit greater sensitivity and specificity than those prepared with the RINe values > 2.3. These findings suggest that the DV200 is superior to the RINe, especially for low‐quality RNA, because it is a more consistent assessment of the amount of the 1 st NGS library product per input.
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DV200 analysis has proven to be a sufficient RNA quality index, with a stronger correlation with the amount of library product than the RNA integrity number for FFPE samples.
“DV200 analysis measures the percentage of RNA fragments which are > 200 nucleotides in length within a sample, and has proven to be a sufficient RNA quality index, with a stronger correlation with the amount of library product, than the RNA integrity number for FFPE samples. 18”
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- Authors
- Matsubara, T., Soh, J., Morita, M., Uwabo, T., Tomida, S., Fujiwara, T., Kanazawa, S., Toyooka, S., & Hirasawa, A.
- Affiliations
- Okayama University Hospital (JP)
- Okayama University (JP)
- Corresponding author
- Soh, Junichi
- ORCID
- Venue
- BioMed Research International
- Publisher
- Wiley
- Published
- 2020-01
- Volume
- 2020
- Issue
- 1
- Pages
- 9349132-9349132
- ISSN
- 1110-7243 (ISSN-L)
- 2314-6133
- 2314-6141
- PMC
- PMC7063185
- OpenAlex
- W3007740300
- Cited by
- 97
- References
- 14
- Citations by year
- 2026: 20 · 2025: 20 · 2024: 22 · 2023: 15 · 2022: 7 · 2021: 12 · 2020: 1
- FWCI
- 2.84
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- BioMed Research International (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Institutional Repositories DataBase (IRDB) (submittedVersion, cc-by)
- Okayama University Scientific Achievement Repository (Okayama University) (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Japan Agency for Medical Research and Development
- Japan Society for the Promotion of Science (15H04830, 16K19454, 16K10681)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene expression and cancer classification
- Keywords
- RNA integrity, RNA quality assessment, formalin-fixed paraffin-embedded tissue, fresh frozen tissue, cell lines
- MeSH terms
- Humans, RNA, Sensitivity and Specificity, Gene Library, Paraffin Embedding, High-Throughput Nucleotide Sequencing
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