Ribodepletion is a laboratory method for removing ribosomal RNA (rRNA) from a total RNA sample. This technique enriches the sample for messenger RNA (mRNA) and non-coding RNAs (ncRNAs), which hold more direct information about a cell’s gene activity. The process acts as a specific filter, isolating these relevant transcripts from the abundant rRNA. This preparatory step is used before genetic analyses that examine the complete set of RNA transcripts to understand cellular function, development, and disease.
The Challenge of Ribosomal RNA Abundance
The total RNA content within a cell is overwhelmingly composed of ribosomal RNA, accounting for 80% to 95% of all RNA molecules. The role of rRNA is structural, as it forms the building blocks of ribosomes, the cellular factories that synthesize proteins. Because ribosomes are constantly needed, cells maintain a vast supply of rRNA. This high quantity presents a challenge for certain types of genetic analysis.
This abundance is an issue for sequencing-based research, as the volume of rRNA can obscure signals from rarer RNA molecules. For example, messenger RNA, which reflects active genes, may only constitute 1-5% of the total RNA. Without removing rRNA, analytical resources would be wasted sequencing it instead of the transcripts that reveal dynamic cellular processes.
The sequences of rRNA are largely constant and provide little insight into the regulatory changes that occur during disease or in response to stimuli. Researchers are more interested in the transcriptome, which includes the mRNA and ncRNA transcripts that reflect cellular behavior. Depleting rRNA allows the analysis to focus on these molecules, which offer a more dynamic picture of gene expression.
Mechanisms of Ribodepletion
Ribodepletion relies on the specific pairing of nucleic acid sequences. The process uses short, synthetic DNA strands, known as probes, with sequences complementary to the cell’s rRNA. During hybridization, the total RNA sample is mixed with these probes. The probes bind exclusively to their rRNA targets, forming stable DNA-RNA hybrid molecules and tagging the unwanted rRNA for removal.
Once the rRNA is tagged, it is physically separated from the sample. The most common method uses magnetic beads. The DNA probes are engineered with a biotin molecule attached to one end. After hybridization, streptavidin-coated magnetic beads are added, which attach firmly to the biotin on the probe-rRNA complexes due to the strong and specific bond between biotin and streptavidin.
With the rRNA linked to magnetic beads, the sample tube is placed in a magnetic rack, which pulls the beads and attached rRNA to the side. The remaining solution, containing the enriched mRNA and ncRNA, is then carefully pipetted out. This method efficiently purifies the sample by removing the ribosomal RNA. An alternative approach uses an enzyme like RNase H, which recognizes and degrades the RNA within the DNA-RNA hybrid, destroying the rRNA while leaving other molecules intact.
Impact on Gene Expression Analysis
Ribodepletion is a preparatory step for whole-transcriptome sequencing, an analysis known as RNA-Seq. By removing abundant rRNA, the technique allows for a deeper survey of all RNA transcripts in a cell. This enhanced view helps detect low-quantity mRNA transcripts. It also enables the discovery and analysis of various non-coding RNAs not captured by other methods.
The method is valuable for studying transcripts that lack a poly(A) tail. While most mature mRNAs have this tail, many regulatory molecules like long non-coding RNAs (lncRNAs) and circular RNAs do not. Additionally, the poly(A) tails on mRNA are often lost in degraded RNA samples, such as those from archived tissues. Ribodepletion provides a reliable way to prepare these types of samples for sequencing.
This comprehensive approach leads to more accurate measurements of gene expression levels across the genome. By allowing researchers to identify novel transcripts, the detailed information gathered from ribodepleted samples helps advance the understanding of cellular function, organism development, and the molecular basis of diseases.
Alternatives to Ribodepletion
The primary alternative to ribodepletion is poly(A) selection, which uses positive selection instead of depletion. This technique uses oligo(dT) probes, which are short DNA strands made of repeating thymine bases. These probes bind specifically to the poly(A) tail found on the end of most mature messenger RNAs in eukaryotic organisms.
The poly(A) selection process isolates mRNA by capturing it, often on magnetic beads coated with oligo(dT) probes. All other RNA types that lack a poly(A) tail, including rRNA and most non-coding RNAs, are washed away. This approach is efficient and cost-effective for studying protein-coding genes in high-quality, intact RNA samples.
The choice between these methods depends on the experiment’s goals and the sample’s condition. Poly(A) selection is preferred for standard gene expression profiling of coding RNAs. Ribodepletion is necessary when the research must include non-polyadenylated RNAs or when analyzing RNA from bacteria, which lack poly(A) tails. It is also the superior method for degraded samples where the poly(A) tails may be lost.