An alternative to depleting rRNA sequences prior to cDNA library synthesis is to apply cDNA normalization (also called Cot filtration) approaches that remove highly abundant sequences from cDNA libraries. In normalization, double-stranded DNA (dsDNA) populations are first denatured and then allowed to re-anneal at an elevated temperature. Highly abundant sequences hybridize at higher rates (proportional to the square of their concentration) and, if the re-annealing reaction is stopped at a suitable time point (e.g., 4–24 h), these will comprise the majority of double-stranded species. If double-stranded and single-stranded cDNA can then be separated, representation of the highest abundance species in the resulting ss fraction can be significantly reduced. The two common approaches for separating ss-cDNA and ds-cDNA populations include enzymatic digestion of ds-cDNA using a duplex specific nuclease (DSN) and physical separation of ds-cDNA from ss-cDNA through methods such as hydroxyapatite chromatography (HAC).
Scientists at Sandia National Laboratories have developed a micro-column based HAC approach for normalization using convenient re-packable cartridges that is rapid, reproducible, and amenable to future automated sample preparation platforms . They present a comparison of our microcolumn HAC-based method with a commercial rRNA-depletion kit, Ribo-Zero, and a DSN normalization kit for normalizing SGS libraries prepared from Escherichia coli K-12 or human peripheral blood mononuclear cell (PBMC) total RNA, respectively. Sequencing of RNA-seq cDNA libraries followed by alignment to either the E. coli K-12 or human (hg19) genome was used to measure rRNA abundance, non-rRNA transcript enrichment, and in the case of E. coli K-12, coverage across the entire bacterial transcriptome. Microcolumn HAC-based normalization proved to be an effective, cost saving alternative to commercial Ribo-Zero and DSN normalization kits, and the first step toward a fully automated system incorporating HAC normalization into RNA-seq cDNA library preparation workflows.
- Vandernoot VA, Langevin SA, Solberg OD, Lane PD, Curtis DJ, Bent ZW, Williams KP, Patel KD, Schoeniger JS, Branda SS, Lane TW. (2012) cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications. Biotechniques 53(6), 373-80.[article]