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 (8) + Submit your own case

severe
No Depletion Due to Incorrect Probe Design Input
Percentage of reads mapping to the targeted sequence does not decrease after depletion treatment. Sequencing analysis shows target RNA remains at original levels.
💡 4 · ✓ 5
severe
Probe Integrity Compromised During Storage or Synthesis
Target RNA depletion efficiency is significantly reduced or absent. Sequencing shows minimal reduction in targeted reads despite proper protocol execution.
💡 3 · ✓ 4
severe
DNA Contamination Interferes with RNA Quantification and Depletion
RNA depletion shows poor efficiency with high residual target reads. RNA quantification may be inaccurate and inconsistent with expected values.
💡 3 · ✓ 4
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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