rRNA is the predominant form of RNA found in cells, often exceeding 80%. In human cells, mRNA accounts for only 1–5% of the total cellular RNA, although the actual amount varies depending on the cell type and physiological state. Interestingly, some mRNAs constitute up to 3% of the mRNA pool, while others account for less than 0.1%. Due to the differences between abundancy of rRNA and other RNA species, reads mapping to rRNA dominate the dataset during sequencing. This dominance hampers the detection of low-abundance transcripts and distorts gene expression profiles.

Ribodepletion

Ribodepletion refers to the targeted removal of rRNA from RNA samples before sequencing. By depleting rRNA, researchers improve the detection of low-level transcripts, facilitate differential expression analysis and minimize false positives and negatives. 

Below we present three recent examples offering more details on how ribodepletion impacts gene expression analysis:

A study using RNA from fresh frozen solid tumor samples compared different methods of analysis including microarray, poly(A) enrichment followed by RNA sequencing, ribodepletion followed by RNA sequencing and Duplex Specific Nuclease Sequencing (DSN-Seq). In fresh samples poly(A) enrichment and ribodepletion provided equivalent rRNA removal efficiency, coverage uniformity, genome-based mapped reads, and reduced 5′- to- 3′ bias. In addition, ribodepletion produced highly consistent quantification of transcripts when compared with microarray, and substantially more information on non-poly(A) RNA. 

In a second study researchers were performing single cell RNA sequencing in planarians. To maximize retrieval of protein-coding transcripts, most scRNA-seq methods capture mRNA by poly(A) enrichment. However, some transcripts such as 16S rRNA from mitochondrial genes, can escape this elimination and overwhelm libraries. Using a CRISPR/Cas9-based approach to remove 16S ribosomal rRNA produced a substantial increase in the number of genes detected per cell, reduced dropout rates, retrieved more clusters, and revealed more differentially expressed genes compared to in silico depletion.

A final study focused on the transcription profile of a marine sponge, holobiont, looking at the mRNA levels of both the host and symbiotic bacteria. Traditionally, poly(A)-enriched libraries have been used to capture eukaryotic mRNA, but the ability of this method to adequately capture bacterial mRNAs is unclear because of the short half-life of the bacterial transcripts. Different methods were compared, and it was found that rRNA depletion in both host and symbionts in a single step produced a good representation of the community and constituted a time and cost-efficient approach that can be applied to any system comprising a eukaryotic host and bacterial symbionts.

Conclusion

Ribodepletion methods enhance gene expression studies by selectively removing unwanted transcripts, improving library complexity, and accurately quantifying expression levels. Revvity offers different solutions based on hybridization-probe approach or CRISPR-Cas9 system to remove uninformative molecules in single cell sequencing, whole blood or even complex communities containing host and bacteria RNA. 

For research use only. Not for use in diagnostic procedures.

References:

1. Zhao, W., He, X., Hoadley, K.A. et al. Comparison of RNA-Seq by poly (A) capture, ribosomal RNA depletion, and DNA microarray for expression profiling. BMC Genomics 15, 419 (2014).
2. Wang, KT., Adler, C.E. CRISPR/Cas9-based depletion of 16S ribosomal RNA improves library complexity of single-cell RNA-sequencing in planarians. BMC Genomics 24, 625 (2023).
3. Xiang, X., Poli, D., Degnan, B.M. et al. Ribosomal RNA-Depletion Provides an Efficient Method for Successful Dual RNA-Seq Expression Profiling of a Marine Sponge Holobiont. Mar Biotechnol 24, 722–732 (2022).