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RNA DepletionFree in-browser calculator

RNA Depletion Probe Designer.

Design tiled antisense probes for rRNA or custom RNA depletion. Paste target sequences in FASTA format, set probe length and tiling overlap, then cluster to remove redundant probes. Exports probe FASTA, IDT order CSV, and design parameters. All computation runs client-side.

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Validated2026-04-07
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Load example RNA Depletion Probe Designer data to see the full workflow

Target Sequences (FASTA)

Paste one or more target RNA/DNA sequences in FASTA format. Probes will be designed as antisense (reverse-complement) oligos tiled across each target. U bases are automatically converted to T.

Design Parameters

15\u2013150 nt (default 50)

0 = abutting (no overlap). Higher values increase coverage redundancy.

100% = exact-match only. Lower = more aggressive deduplication.

When to use

  • Design antisense probes for rRNA depletion before RNA-seq library preparation
  • Create custom depletion panels for any target RNA (globin, mitochondrial, viral)
  • Tile probes across multiple related sequences and cluster to remove redundant oligos
  • Generate IDT-ready order files for oligo synthesis

Do not use for

  • Designing capture/enrichment probes (positive selection) — those require different length and density optimization
  • qPCR primer design — use a dedicated primer design tool
  • CRISPR guide RNA design — use our sgRNA Template Oligo Designer instead

Use overlapping tiles for structured targets

rRNA has extensive secondary structure. Overlapping probes (10–25 nt overlap) ensure that even regions partially occluded by structure are covered by at least one probe that can access the target.

Monitor Tm uniformity

A probe set with Tm range > 15°C will hybridize unevenly at a single annealing temperature. Consider splitting probes into Tm-matched pools or adjusting probe length to narrow the range.

Order DNA probes, not RNA

Depletion probes should be ordered as DNA oligos, not RNA. RNase H specifically cleaves the RNA strand of RNA:DNA hybrids, so DNA probes are essential for the mechanism to work.

Validate depletion efficiency by qPCR

After depletion, use qPCR with rRNA-specific primers to quantify depletion efficiency before committing to sequencing. Expect > 95% rRNA removal for a well-designed probe set.

Consider probe concentration carefully

Probe-to-target molar ratio affects depletion efficiency. Too little probe leaves rRNA behind; too much probe can non-specifically trap other transcripts. Follow kit recommendations (typically 10–100x molar excess).

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Method

The tool tiles probes of user-specified length across each target sequence with configurable overlap. Probes are the reverse-complement (antisense) of the target region. After tiling, probes are clustered by sequence identity using a greedy algorithm similar to CD-HIT: probes above the identity threshold are collapsed to a single representative. Tm is calculated using the SantaLucia (1998) nearest-neighbour method with salt correction. GC and Tm distribution histograms are computed for quality assessment.

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Validated

Last validated 2026-04-07. Calculations are designed for planning and documentation support; verify procurement decisions against manufacturer specifications or institutional SOPs.

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How to cite

How to Cite

ConductScience RNA Depletion Probe Designer (v1.24.0). ConductScience, Inc. 2026. Available at: https://conductscience.com/tools/rna-depletion-probe-designer

Morlan JD et al. Selective Depletion of rRNA Enables Whole Transcriptome Profiling of Archival Fixed Tissue. PLoS ONE. 2012;7:e42882. doi:10.1371/journal.pone.0042882

Adiconis X et al. Comparative analysis of RNA sequencing methods for degraded or low-input samples. Nature Methods. 2013;10:623-629. doi:10.1038/nmeth.2483

SantaLucia J Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. PNAS. 1998;95:1460-1465. doi:10.1073/pnas.95.4.1460

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.

Applications beyond rRNA depletion

While rRNA depletion is the most common use case, tiled antisense probe panels can be designed for:

  • Globin mRNA depletion: Removing highly abundant alpha- and beta-globin transcripts from blood RNA samples.
  • Mitochondrial RNA depletion: Reducing mt-rRNA reads that dominate single-cell RNA-seq libraries.
  • Custom transcript depletion: Removing any unwanted abundant transcript (e.g., transgene, viral RNA) to enrich for targets of interest.
  • DASH (Depletion of Abundant Sequences by Hybridization): Cas9-based depletion using guide RNAs designed with similar tiling principles.

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