How RNA depletion works
RNA depletion uses antisense oligonucleotide probes to selectively remove unwanted RNA species (typically rRNA) from a total RNA sample.
The basic workflow:
1. Probe hybridization: Antisense DNA probes complementary to target rRNA are mixed with total RNA and heated to denature secondary structures, then slowly cooled to allow probe:target annealing.
2. RNase H digestion: RNase H specifically cleaves the RNA strand of RNA:DNA hybrids, fragmenting the targeted rRNA while leaving non-targeted RNA intact.
3. Cleanup: Degraded rRNA fragments and free probes are removed by DNase treatment followed by bead-based size selection or column purification.
4. Library preparation: The enriched RNA is then used for standard RNA-seq library preparation.
Principles of depletion probe design
Tiling strategy:
Probes are tiled across the target RNA to ensure complete coverage. Gaps between probes leave rRNA fragments that escape depletion and consume sequencing reads.
Probe length trade-offs:
- Shorter probes (25–40 nt): More uniform Tm, easier to synthesize, but may have lower binding efficiency and be displaced by RNA secondary structure.
- Longer probes (50–80 nt): Stronger hybridization, better displacement of secondary structure, but wider Tm variation and higher synthesis cost.
GC content and Tm:
Ideal probes have 40–60% GC content and uniform Tm. Probes with extreme GC (<30% or >70%) may hybridize poorly. A narrow Tm range across the probe set ensures uniform hybridization at a single annealing temperature.
Cross-species panels:
For samples with mixed-species rRNA (e.g., host + pathogen), design probes against all relevant rRNA sequences and cluster to remove redundant probes that would target conserved regions of both.
