title: RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Specimens for Gene Expression Quantification and Data Mining.
abstract: 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.
authors: Department of Cancer Biology, Vanderbilt University, Nashville, TN, USA.
authors: Yan
authors: 0000-0001-5252-3960
authors: Y
authors: Guo
authors: Division of Epidemiology, Department of Medicine, Vanderbilt Epidemiology Center and Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USA.
authors: Jie
authors: J
authors: Wu
authors: Department of Cancer Biology, Vanderbilt University, Nashville, TN, USA.
authors: Shilin
authors: 0000-0002-3787-8111
authors: S
authors: Zhao
authors: Department of Biostatistics, Vanderbilt University, Nashville, TN, USA.
authors: Fei
authors: F
authors: Ye
authors: Division of Epidemiology, Department of Medicine, Vanderbilt Epidemiology Center and Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USA.
authors: Yinghao
authors: Y
authors: Su
authors: Genentech, Baltimore, MD, USA.
authors: Travis
authors: T
authors: Clark
authors: Department of Cancer Biology, Vanderbilt University, Nashville, TN, USA.
authors: Quanhu
authors: 0000-0001-8951-9295
authors: Q
authors: Sheng
authors: Department of Biochemistry, Vanderbilt University, Nashville, TN, USA.
authors: Brian
authors: 0000-0002-4269-2274
authors: B
authors: Lehmann
authors: Division of Epidemiology, Department of Medicine, Vanderbilt Epidemiology Center and Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USA.
authors: Xiao-Ou
authors: XO
authors: Shu
authors: Division of Epidemiology, Department of Medicine, Vanderbilt Epidemiology Center and Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USA.
authors: Qiuyin
authors: 0000-0002-9384-5648
authors: Q
authors: Cai