Improvements to RiboMinus™ Eukaryote rRNA Depletion Probe Design and Functionality to Enable a Faster and More Complete Workflow

• Published in: Journal of biomolecular techniques 2013-05, Vol.24 (Suppl), p.S72-S72
• Main Authors: Qu, Luming, Hernandez, Natalie Supunpong, Chapman, Laura, Burnett, Chris, Gu, Jian, Bramlett, Kelli, Schageman, Jeff, Brockman, Joel, Hinahon, Charmaine
• Format: Article
• Language: English
• Subjects: Poster Session Abstracts
• ISSN: 1524-0215; 1943-4731

Cellular RNA is composed mainly of cytoplasmic and mitochondrial ribosomal RNA (rRNA). Since rRNAs are not usually the target of whole transcriptome RNA-Seq studies and can potentially take up a majority of valuable sequencing reads, fractionation of the total RNA to obtain rRNA depleted RNA is a necessary first step. The current RiboMinus™ design has had limited success with partially degraded total RNA, due to an abundance of fragmented rRNA contamination in compromised RNA that the current design of probes do not address. We have made improvements to the design and functionality of the RiboMinus™ Eukaryote Kit for RNA-Seq and have expanded its utility to include rRNA depletion from partially degraded total RNA samples. Improvements include: 1. increased the number of rRNA probes; 2. expanded the probe design to include mitochondrial as well as cytoplasmic rRNA; 3. optimized the streptavidin-biotin hybridization time to bring the overall workflow time to about one hour; 4. developed a bead based concentration step enabling scalability of the protocol to multiple samples. These improvements enable the new RiboMinus™ Eukaryote System v2 to be a competitive option for rRNA depletion upstream of Ion Total RNA-Seq Kit v2 library preparation while preserving the whole transcriptome population including RNA transcripts less than 200 nt in length. This workflow enables discovery of new transcripts and accurate gene expression profiling of biologically relevant RNA species beyond those of polyadenylated mRNA.