Artistic rendering of RNA in a mountain landscape

What is ribosomal RNA depletion?

Ribosomal depletion is a critical method in transcriptomics that allows for efficient detection of functionally relevant coding as well as non-coding transcripts through removal of highly abundant rRNA species. Enrichment of whole transcriptome RNA by ribosomal RNA depletion (rRNA depletion) is a critical step in preparing samples for use in gene expression microarrays, RNA-Seq, and other analysis methods. The regulation of expression and the types and functions of RNA are far more complex than was previously thought, and tools to study RNA have developed to keep pace with the interest.

rRNA depletion vs poly-A selection

Enrichment of mRNA by capturing the polyadenylated 3′ end of the transcripts is routine in many RNA sequencing preparation workflows. This method is rapid and specific for poly-A RNA. However, if the RNA has degraded to the point where most of the mRNA is separated from the 3′ tail, or you need to isolate non-polyadenylated transcripts such as long non-coding RNAs, rRNA depletion may be a better option. Ribosomal RNA depletion methods directly bind and remove rRNA based on ribosomal RNA sequences, leaving other transcripts in the sample regardless of length or poly-A content.

Three cell diagrams that compare methods to select eukaryotic RNA by showing the selected species of RNA outside of the cell.

Figure 1. Methods for selecting RNA species of interest. (A) Total RNA content in eukaryote cell, (B) selection of mRNA content in a eukaryote cell by oligo-dT binding using Dynabeads mRNA DIRECT Micro kit, and (C) complete transcriptome isolation with the Ribominus Eukaryote v2 kit for rRNA depletion.

Ribosomal RNA depletion promotes efficient detection of functionally relevant coding as well as non-coding transcripts through removal of highly abundant rRNA species.

Why do rRNA depletion for RNA-seq?

Ribosomal RNA depletion increases relative abundance of non-poly(A)-tailed RNA, preprocessed RNA, small tRNAs, siRNA, snRNA, microRNA, fragmented mRNA, and other regulatory RNAs that cannot be purified via polyA+ selection methods.

How probe-based depletion of ribosomal RNA can improve RNA-seq results

In eukaryotic cells, there are about 10x more molecules of ribosomal RNA than all other types of RNA combined. For transcriptomics, it is this non-ribosomal RNA that needs to be sequenced. If total RNA is sequenced, about 90% of RNA-seq reads will be ribosomal, leaving only ~10% of reads relevant to your experiment. If rRNA is 97% depleted, the fraction of relevant reads increases to almost 80%. By achieving 99% rRNA depletion, for RNA-seq about 90% of the reads will be of the transcriptome.

Illustration of theoretical NGS reads in which 0%, 97%, or 99% of rRNA is depleted from the total RNA sample

Figure 2. How rRNA % depletion translates to NGS % mapped reads.

Ribosomal RNA depletion probe technology for RNA-seq or microarrays

RiboMinus rRNA depletion technology is designed to enrich the whole spectrum of RNA transcripts by selectively depleting ribosomal RNA molecules (rRNA), regardless of their polyadenylation status or the presence of a 5'-cap structure. The RiboMinus rRNA depletion method has been shown to remove the vast majority of the most abundant ribosomal RNA molecules (up to 99.9%) to allow for greater interrogation of less abundant transcripts, giving you more useful data from your RNA-seq or microarray analyses.

How does rRNA depletion work?

Biotinylated probe oligonucleotides designed to bind ribosomal RNA are added to a sample of total RNA and allowed to hybridize. Streptavidin-coated paramagnetic beads are then added to the sample, capturing biotin-oligo-rRNA complexes. A magnet is then applied, allowing easy removal of bead-captured rRNA from the total RNA sample. The ribosomal RNA-depleted sample may then be concentrated depending on the downstream application. Ribominus rRNA depletion kits provide species-specific probes, Dynabeads streptavidin-coated magnetic beads and concentrating spin columns (optional) to streamline your ribosomal RNA depletion workflow.

Successful rRNA depletion starts with high-quality total RNA

To help ensure success of your transcriptomics experiments, make sure your starting material is of good quality. Explore these solutions for total RNA isolation.

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