Home › Genetics / Genomics › The Importance of Performing Ribosomal RNA Depletion
Steps
  1. 1 Understand ribosomal RNA abundance problem 00:05
  2. 2 Compare mRNA pull-down vs rRNA depletion 00:37
  3. 3 Apply enzymatic rRNA depletion workflow 01:07
  4. 4 Assess benefits of direct depletion method 01:40
  5. 5 Customize protocol for non-standard organisms 02:11
  6. 6 Design organism-specific oligonucleotides 02:48
  7. 7 Validate efficient rRNA depletion results 03:14
Genetics / Genomics RocheSequencingUSA

The Importance of Performing Ribosomal RNA Depletion

Protocol
Difficulty
intermediate

Steps

1
Understand ribosomal RNA abundance problem

Learn that ribosomal RNA comprises over 90% of total cellular RNA, wasting sequencing resources and reducing coverage of transcripts of interest. The goal is to remove rRNA to improve sequencing efficiency and data quality.

▶ 00:05
2
Compare mRNA pull-down vs rRNA depletion

Examine bead-based pull-down methods that selectively capture polyadenylated mRNA species, which work well for high-quality RNA but have limitations with degraded samples like FFPE tissue. Understand why alternative depletion approaches are needed.

▶ 00:37
3
Apply enzymatic rRNA depletion workflow

Use the Kappa RiboErase workflow which employs designed complementary DNA oligonucleotides that hybridize to rRNA species and are then directly depleted using RNase H enzyme.

▶ 01:07
4
Assess benefits of direct depletion method

Recognize that enzymatic rRNA depletion retains non-coding RNAs and precursor mRNA species, enabling a more complete transcriptome analysis compared to mRNA pull-down approaches. This method is also more efficient and suitable for low-input and degraded samples.

▶ 01:40
5
Customize protocol for non-standard organisms

Recognize that standard kits are designed for human, mouse, and rat species, requiring customized oligonucleotide design for other organisms like bacteria, yeast, or plants where standard oligos are not homologous to target RNA.

▶ 02:11
6
Design organism-specific oligonucleotides

Collaborate with customers to design custom DNA oligonucleotides tailored to specific bacterial or other non-standard RNA targets, enabling seamless integration into the existing enzymatic depletion workflow.

▶ 02:48
7
Validate efficient rRNA depletion results

Confirm that custom oligonucleotides with minimal optimization achieve highly efficient rRNA depletion when processed through the standard workflow, demonstrating successful protocol adaptation for diverse organisms.

▶ 03:14

🚨 Failure Case Library (9) + 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
Genome Annotation Version Mismatch Between Design and Analysis
Target sequence used to design probes differs from the reference used to evaluate depletion. Depletion appears ineffective when analyzed against current genome annotations.
💡 4 · ✓ 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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