Home › Molecular Biology › rRNA depletion strategies
Steps
  1. 1 Understand the rRNA abundance problem --:--
  2. 2 Design DNA probes complementary to rRNA 03:29
  3. 3 Apply RNase H digestion method 05:13
  4. 4 Attach biotinylated probes to magnetic beads 06:31
  5. 5 Equilibrate beads and incubate with sample 08:07
  6. 6 Isolate rRNA-depleted supernatant using magnet 08:29
  7. 7 Overview of commercial rRNA depletion kits 09:40
Molecular Biology the bumbling biochemist

rRNA depletion strategies

Protocol
Difficulty
intermediate

Steps

1
Understand the rRNA abundance problem

Learn that ribosomal RNA comprises 80-90% of cellular RNA, which interferes with studying messenger RNA and other RNA species. Understand why rRNA depletion is necessary for accurate gene expression analysis through mRNA sequencing or ribosome profiling.

▶ --:--
2
Design DNA probes complementary to rRNA

Create or purchase short DNA oligonucleotides (oligos) that specifically complement rRNA sequences. These probes form base-pairing interactions with target rRNA molecules to enable selective depletion.

▶ 03:29
3
Apply RNase H digestion method

Use DNA probes to form RNA-DNA hybrids with rRNA, then add RNase H enzyme to selectively cut and degrade the RNA portion. This degrades the targeted rRNA so it cannot be sequenced.

▶ 05:13
4
Attach biotinylated probes to magnetic beads

Add biotinylated DNA oligos complementary to rRNA sequences to your sample. The biotin labels bind tightly to streptavidin-conjugated magnetic beads, creating a capture system for rRNA removal.

▶ 06:31
5
Equilibrate beads and incubate with sample

Pre-equilibrate magnetic beads with appropriate buffer, then add your RNA sample and incubate for approximately 15 minutes with gentle mixing on a shaker. This allows probes to bind rRNA and beads to capture the complexes.

▶ 08:07
6
Isolate rRNA-depleted supernatant using magnet

Place the bead-sample mixture on a magnetic platform to pull rRNA-bound beads to the magnet wall. Carefully pipette the clear supernatant containing rRNA-depleted sequences into a new tube for downstream library preparation.

▶ 08:29
7
Overview of commercial rRNA depletion kits

Review available commercial kits using biotin-streptavidin pulldown (Illumina Ribo-Zero, Lexogen) and RNase H-based methods (NEB, Takara). Discuss SPRI bead purification as an alternative for temporary DNA/RNA precipitation and cleanup.

▶ 09:40

🚨 Failure Case Library (6) + Submit your own case

moderate
Chemical Contaminants Inhibit Probe Hybridization
Depletion efficiency is reduced or completely absent. Target RNA remains at high levels after treatment despite using validated probe designs.
💡 4 · ✓ 5
moderate
Non-Uniform Depletion Across Targeted Sequences
Depletion efficiency varies significantly across different targeted sequences. Some target regions show good depletion while others remain at high levels.
💡 4 · ✓ 5
moderate
Bioanalyzer peaks below 85 bp detected
Presence of Bioanalyzer peaks smaller than 85 bp observed after PCR cleanup. These peaks represent residual primers from the amplification reaction.
💡 3 · ✓ 4
moderate
Additional high molecular weight peak at ~1,000 bp
Bioanalyzer shows an additional peak at higher molecular weight than expected library size (approximately 1,000 bp). This represents single-stranded library products that have self-annealed to form heteroduplexes.
💡 5 · ✓ 5
moderate
Adaptor-dimer peak at approximately 127 bp
Bioanalyzer shows a distinct peak at approximately 127 bp representing adaptor-dimer formation. These dimers will cluster and be sequenced, potentially consuming sequencing capacity.
💡 5 · ✓ 5
moderate
Broad library size distribution on Bioanalyzer
Library shows a broad size distribution on Bioanalyzer with longer insert sizes than expected. This indicates heterogeneous fragment lengths in the final library.
💡 4 · ✓ 5
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