RNA sequencing, or RNA-seq, is a powerful technique that allows researchers to study the complete set of RNA molecules, known as the transcriptome, present in a cell, tissue, or organism at a specific time. This method provides detailed insights into gene activity and regulation. To optimize the data obtained from these experiments, ribosomal RNA (rRNA) depletion is a common preparatory step for many RNA-seq workflows.
The Problem with Ribosomal RNA
Ribosomal RNA (rRNA) presents a significant challenge for RNA-seq experiments due to its overwhelming abundance within a total RNA sample. In typical eukaryotic cells, rRNA can constitute 80-90% or more of the total RNA content. This high proportion means that if total RNA is sequenced without any pre-processing, a substantial majority of the sequencing reads would correspond to rRNA.
Sequencing such a large quantity of rRNA is not informative for studying gene expression, as it primarily functions as a structural component of ribosomes and does not directly carry genetic information for protein coding. Its excessive presence can “swamp” sequencing data, making it difficult and expensive to obtain sufficient reads for less abundant, biologically relevant transcripts like messenger RNA (mRNA) or various non-coding RNAs.
How rRNA Depletion Works
rRNA depletion methods involve using probes designed to specifically bind to and remove rRNA molecules from the sample. One common approach utilizes biotinylated oligonucleotide probes that are complementary to rRNA sequences. These probes hybridize to the rRNA molecules in the total RNA sample.
Following hybridization, streptavidin-coated magnetic beads are introduced to the mixture. Streptavidin has a strong affinity for biotin, so the biotinylated probes, now bound to rRNA, are captured by these magnetic beads. A magnet is then applied to the sample tube, pulling the beads, along with the bound rRNA, to the side of the tube, allowing the rRNA-depleted RNA solution to be collected. Another method involves RNase H, an enzyme that specifically degrades RNA within an RNA-DNA hybrid, where DNA probes bind to rRNA. Commercial kits are available for various sample types.
Advantages and Key Applications
rRNA depletion offers several advantages for RNA-seq experiments by allowing for a more focused and comprehensive analysis of the transcriptome. By removing the highly abundant rRNA, sequencing depth can be directed towards less abundant transcripts, thereby increasing the sensitivity to detect genes with low expression levels. This enhanced sensitivity is beneficial for uncovering rare transcripts with important biological roles.
rRNA depletion enables the capture of a broader range of RNA types beyond just messenger RNA (mRNA). This includes various non-coding RNAs (ncRNAs) such as long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs, which may not have poly(A) tails and are often of significant biological interest in gene regulation. The method is also valuable for analyzing samples with degraded RNA, where poly(A) tails may be compromised or lost, making poly(A) selection ineffective. For instance, it is useful for studying microbial communities, as bacterial mRNA lacks a poly(A) tail, or for analyzing clinical samples like formalin-fixed paraffin-embedded (FFPE) tissues.
When to Choose rRNA Depletion
Choosing between rRNA depletion and poly(A) selection depends on the specific goals of the RNA-seq experiment. Poly(A) selection primarily targets messenger RNA (mRNA) because most eukaryotic mRNA molecules possess a poly(A) tail at their 3′ end, allowing for their isolation using oligo-dT primers attached to magnetic beads. This method is simpler and more cost-effective if the research focuses exclusively on intact, polyadenylated mRNA.
Conversely, rRNA depletion is the preferred method in several scenarios. It is ideal when studying non-coding RNAs, which lack poly(A) tails, or when working with prokaryotic samples (e.g., bacteria, archaea) whose mRNA lack poly(A) tails. Additionally, if RNA samples are degraded or compromised, rRNA depletion is more suitable as it does not rely on the integrity of the poly(A) tail. While rRNA depletion can lead to a higher percentage of non-coding reads and may require greater sequencing depth compared to poly(A) selection for the same exonic coverage, it offers greater flexibility for diverse and challenging sample types.