RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Specimens for Gene Expression Quantification and Data Mining
Background. Proper rRNA depletion is crucial for the successful utilization of FFPE specimens when studying gene expression. We performed a study to evaluate two major rRNA depletion methods: Ribo-Zero and RNase H. RNAs extracted from 4 samples were treated with the two rRNA depletion methods in dup...
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| Format: | Article |
| Language: | English |
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Wiley
2016-01-01
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| Series: | International Journal of Genomics |
| Online Access: | http://dx.doi.org/10.1155/2016/9837310 |
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| author | Yan Guo Jie Wu Shilin Zhao Fei Ye Yinghao Su Travis Clark Quanhu Sheng Brian Lehmann Xiao-ou Shu Qiuyin Cai |
| author_facet | Yan Guo Jie Wu Shilin Zhao Fei Ye Yinghao Su Travis Clark Quanhu Sheng Brian Lehmann Xiao-ou Shu Qiuyin Cai |
| author_sort | Yan Guo |
| collection | DOAJ |
| description | Background. Proper rRNA depletion is crucial for the successful utilization of FFPE specimens when studying gene expression. We performed a study to evaluate two major rRNA depletion methods: Ribo-Zero and RNase H. RNAs extracted from 4 samples were treated with the two rRNA depletion methods in duplicate and sequenced (N=16). We evaluated their reducibility, ability to detect RNA, and ability to molecularly subtype these triple negative breast cancer specimens. Results. Both rRNA depletion methods produced consistent data between the technical replicates. We found that the RNase H method produced higher quality RNAseq data as compared to the Ribo-Zero method. In addition, we evaluated the RNAseq data generated from the FFPE tissue samples for noncoding RNA, including lncRNA, enhancer/super enhancer RNA, and single nucleotide variation (SNV). We found that the RNase H is more suitable for detecting high-quality, noncoding RNAs as compared to the Ribo-Zero and provided more consistent molecular subtype identification between replicates. Unfortunately, neither method produced reliable SNV data. Conclusions. In conclusion, for FFPE specimens, the RNase H rRNA depletion method performed better than the Ribo-Zero. Neither method generates data sufficient for SNV detection. |
| format | Article |
| id | doaj-art-2d1a93de6c514c35a3fca169e8a34726 |
| institution | OA Journals |
| issn | 2314-436X 2314-4378 |
| language | English |
| publishDate | 2016-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | International Journal of Genomics |
| spelling | doaj-art-2d1a93de6c514c35a3fca169e8a347262025-08-20T02:24:14ZengWileyInternational Journal of Genomics2314-436X2314-43782016-01-01201610.1155/2016/98373109837310RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Specimens for Gene Expression Quantification and Data MiningYan Guo0Jie Wu1Shilin Zhao2Fei Ye3Yinghao Su4Travis Clark5Quanhu Sheng6Brian Lehmann7Xiao-ou Shu8Qiuyin Cai9Department of Cancer Biology, Vanderbilt University, Nashville, TN, USADivision of Epidemiology, Department of Medicine, Vanderbilt Epidemiology Center and Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USADepartment of Cancer Biology, Vanderbilt University, Nashville, TN, USADepartment of Biostatistics, Vanderbilt University, Nashville, TN, USADivision of Epidemiology, Department of Medicine, Vanderbilt Epidemiology Center and Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USAGenentech, Baltimore, MD, USADepartment of Cancer Biology, Vanderbilt University, Nashville, TN, USADepartment of Biochemistry, Vanderbilt University, Nashville, TN, USADivision of Epidemiology, Department of Medicine, Vanderbilt Epidemiology Center and Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USADivision of Epidemiology, Department of Medicine, Vanderbilt Epidemiology Center and Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USABackground. Proper rRNA depletion is crucial for the successful utilization of FFPE specimens when studying gene expression. We performed a study to evaluate two major rRNA depletion methods: Ribo-Zero and RNase H. RNAs extracted from 4 samples were treated with the two rRNA depletion methods in duplicate and sequenced (N=16). We evaluated their reducibility, ability to detect RNA, and ability to molecularly subtype these triple negative breast cancer specimens. Results. Both rRNA depletion methods produced consistent data between the technical replicates. We found that the RNase H method produced higher quality RNAseq data as compared to the Ribo-Zero method. In addition, we evaluated the RNAseq data generated from the FFPE tissue samples for noncoding RNA, including lncRNA, enhancer/super enhancer RNA, and single nucleotide variation (SNV). We found that the RNase H is more suitable for detecting high-quality, noncoding RNAs as compared to the Ribo-Zero and provided more consistent molecular subtype identification between replicates. Unfortunately, neither method produced reliable SNV data. Conclusions. In conclusion, for FFPE specimens, the RNase H rRNA depletion method performed better than the Ribo-Zero. Neither method generates data sufficient for SNV detection.http://dx.doi.org/10.1155/2016/9837310 |
| spellingShingle | Yan Guo Jie Wu Shilin Zhao Fei Ye Yinghao Su Travis Clark Quanhu Sheng Brian Lehmann Xiao-ou Shu Qiuyin Cai RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Specimens for Gene Expression Quantification and Data Mining International Journal of Genomics |
| title | RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Specimens for Gene Expression Quantification and Data Mining |
| title_full | RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Specimens for Gene Expression Quantification and Data Mining |
| title_fullStr | RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Specimens for Gene Expression Quantification and Data Mining |
| title_full_unstemmed | RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Specimens for Gene Expression Quantification and Data Mining |
| title_short | RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Specimens for Gene Expression Quantification and Data Mining |
| title_sort | rna sequencing of formalin fixed paraffin embedded specimens for gene expression quantification and data mining |
| url | http://dx.doi.org/10.1155/2016/9837310 |
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